Effect of Lactobacillus plantarum NCU116 Fermentation on Asparagus

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Bioactive Constituents, Metabolites, and Functions

The effect of Lactobacillus plantarum NCU116 fermentation on Asparagus officinalis polysaccharide: Characterization, antioxidative, and immunoregulatory activities Zhi-Hong Zhang, Songtao Fan, Dan-Fei Huang, Qiang Yu, Xiao-Zhen Liu, Chang Li, Sunan Wang, Tao Xiong, Shao-Ping Nie, and Ming-Yong Xie J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b03220 • Publication Date (Web): 25 Sep 2018 Downloaded from http://pubs.acs.org on September 26, 2018

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Journal of Agricultural and Food Chemistry

The effect of Lactobacillus plantarum NCU116 fermentation on Asparagus officinalis polysaccharide: Characterization, antioxidative, and immunoregulatory activities Zhi-Hong Zhang a, Song-Tao Fan a, Dan-Fei Huang a, Qiang Yu a, Xiao-Zhen Liu b, Chang Li a, Sunan Wang c, Tao Xiong a, Shao-Ping Nie a, Ming-Yong Xie *, a a

State Key Laboratory of Food Science and Technology, China-Canada Joint Lab of

Food Science and Technology (Nanchang), Nanchang University, 235 Nanjing East Road, Nanchang 330047, China b

c

Dongguan University of Technology, Dongguan 523808, China Canadian Food and Wine Institute, Niagara College, 135 Taylor Road,

Niagara-on-the-Lake, Ontario L0S 1J0, Canada *

Corresponding author: Professor Ming-Yong Xie, State Key Laboratory of Food

Science and Technology, Nanchang University, 235 Nanjing East Road, Nanchang 330047, China. E-mail address: [email protected] (M. XIE) or [email protected] (M. XIE) Tel & Fax: +86-791-88305860

1

ACS Paragon Plus Environment


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Abstract Lactic acid fermentation represents novel method to produce bioactive functional ingredients, including polysaccharides. In this work, a selected lactic acid bacteria strain NCU116 was used to ferment Asparagus officinalis (asparagus) pulps. Two polysaccharides were subsequently separated from both unprocessed and fermented asparagus pulps, namely asparagus polysaccharide (AOP) and fermented-AOP (F-AOP). The physicochemical and bioactive properties of AOP and F-AOP were characterized and investigated. High-performance anion-exchange chromatography showed that fermentation increased the proportions of rhamnose, galacturonic acid, and glucuronic acid in polysaccharides by 46.70, 114.09 and 12.75‰, respectively. High-performance

size-exclusion chromatography

revealed

that fermentation

decreased the average molecular weight from 181.3 kDa (AOP) to 152.8 kDa (F-AOP). Moreover, the fermentation reduced the particle size and changed the rheology property. In vitro, F-AOP displayed superior free radical scavenging properties than AOP, using DPPH, hydroxyl and superoxide anion radical scavenging assays. In vivo, F-AOP administration dose-dependently promoted a gradual shift from Th17-dominant acute inflammatory response (IL-17 and RORγt) to Th1-dominant defensive immune response (IFN-γ and T-bet). These results indicated that the lactobacillus plantarum NCU116 fermentation was practical and useful to obtain promising bioactive polysaccharides. Keywords: Asparagus officinalis L.; Lactobacillus plantarum NCU116; fermentation; immunoregulatory activity

2


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Table of Contents Graphic

3



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1. Introduction

Lactic acid fermentation represents the easiest and most suitable way for increasing the daily consumption of fresh vegetables and fruits. It offers a natural means to modify technological and nutritional properties of foods and food ingredients

1, 2

. Lactic acid bacteria (LAB) are commonly used among the various

fermentation processes. LAB fermentation changed both profiles and types of bioactive food compounds 3. Being an ancient method of food preservation, LAB fermentation is a well-designed and acknowledged biotechnology for manufacturing functional foods in nowadays 4. LAB-fermented products were frequently associated with enhanced probiotic and/or prebiotic effects 5. For example, Lactobacillus plantarum NCU116 (NCU116) balanced intestinal microbiota disorders in immunosuppression mice 6. The carrot juice fermented with NCU116 ameliorated Type 2 diabetes, accompanied by increased colonic short-chain fatty acids 7. Polysaccharides, rich sources of functional foods and nutrient ingredients, have received extensive attention over the years due to their significant bioactivities, such as anti-tumor

8, 9

, antioxidant

10, 11

, immunomodulatory

12, 13

, antidiabetic activities 14.

Physical and/or chemical processes are the commonly investigated and widely used to alter functionality of native polysaccharides and thus diversify their applications

15

.

Biological alteration of polysaccharides (mainly restricted to enzyme treatments), presents intrinsic high specificity and efficiency, while being rarely reported 16. Being another example of biological alteration, oolong tea after different degrees of fermentation directly influenced their chemical compositions and antioxidant activities 17. Fermentation changed molecular weights of polysaccharides components derived from grape and longan wine

10, 18

. We thus speculated that fermentation

process would be a potential and promising method applied to polysaccharides 4


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treatment and modifications. Asparagus officinalis L. (asparagus) is a perennial dioecious plant and commonly consumed vegetable in many regions of the world

19

. Edible asparagus with an

estimated yield of 760 thousand tons per year, is cultivated in 62 countries worldwide, mainly in Asia, Europe and America

20

. As a well-known healthy and nutritious

vegetable, asparagus is rich in dietary fibre and oligosaccharides

9, 21

. Here, a new

variety of Asparagus named “Jinggang 701”, was chosen as the fermentation substrate for the NCU116. NCU116 was obtained from pickled vegetables in our laboratory, and recognized as a newly identified probiotic with multiple health-promoting properties,

including

immunoregulatory

cholesterol-lowering effects demonstrated antidiabetic

23

6

,

hepatoprotective

22

,

and

. Fermented-products with NCU116 were also

7, 24

. In this study, NCU116 was used to ferment the

asparagus pulps and the fermentation alteration on Asparagus officinalis polysaccharide (AOP) was investigated. Further, the potential bioactivities of AOP and fermented-AOP (F-AOP) were evaluated using in vitro antioxidant system and in vivo immunosuppression model. 2. Materials and methods

2.1 Materials and reagents The "Jinggang 701" green asparagus, cultivated by Jiangxi Academy of Agricultural Sciences, was purchased from Shangrao Zhongde Asparagus Planting Cooperatives (Jiangxi, China). NCU116 was obtained from Nanchang University. The mouse immunoglobulin (Ig) G and IgA kits were obtained from ThermoFisher Scientific (Fair Lawn, NJ, USA). Transcript RT Kit and SYBR Premix Ex TaqTM were purchased from TaKaRa Co. (Shiga, Japan). Cyclophosphamide (CTX) and other reagents were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). 5



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2.2 Preparation of asparagus pulps and fermentation Asparagus was cleaned and cut into 1-3 cm long stems. The asparagus stems were blanched over boiling water for 3 minutes and crushed with Joyoung juicer (JYL-C93T) to get the asparagus pulps. After drying under less than 50 °C, the obtained-asparagus powders were used to isolate AOP. Meanwhile, steriled-asparagus pulps were inoculated with NCU116 (0.5‰, w/w) at 37 °C for 36 hours fermentation, following the second-sterilization to obtain fermented-asparagus pulps. The fermented-asparagus pulps followed above-mentioned drying process to obtain powder materials for F-AOP isolation. 2.3 Polysaccharide extraction Asparagus powders or fermented-asparagus powders were extracted with deionized water (1:20, w/v) 12, the water extract was precipitated with 95% ethanol to produce crude polysaccharides. The crude polysaccharides were deproteinized by Sevage and dialyzed with 8-14 kDa. The dialysate was then concentrated and freeze-dried to obtain refined AOP or F-AOP. 2.4 Characterization of AOP and F-AOP 2.4.1 Monosaccharide composition The monosaccharide compositions of AOP and F-AOP were determined with high-performance

anion-exchange

chromatography

coupled

with

pulsed

amperometric detection (HPAEC-PAD), equipped with a CarboPacTM PA20 (3 mm × 150 mm) column

25

. Briefly, polysaccharides were hydrolyzed by H2SO4 (3.0 mL, 2

M). The neutralized and diluted hydrolysates were filtrated prior to inject into the HPAEC-PAD. The mobile phase consisted of 250 mM NaOH, H2O and 1 M CH3COONa. 2.4.2 Molecular weight 6


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The average molecular weight was measured using high-performance size-exclusion chromatography (HPSEC-MALLS-RID, Wyatt Technology Co., USA) with an OHpak SB-806 HQ column (8.0 mm×300 mm, Shodex Co., Japan) 25. The mobile phase: 0.1 M NaNO3; the flow rate: 0.5 mL/min; injection volume: 50.0 µL; and chromatogram was analyzed using ARTRAV software (Wyatt Technology Co., USA). 2.4.3 Apparent viscosity The apparent viscosity was measured at 25 °C by ARES-G2 Rheometer (TA Co., USA), equipped with a 42 mm cone plate. The effects of shear rate and concentration on the viscosity of AOP and F-AOP solution were evaluated. 2.4.4 Scanning electron microscopy analysis The AOP and F-AOP samples were prepared by sticking them to one side of double-sided adhesive tape attached to a circular specimen stub, and sputter coated with vacuum spray gold. A thermal field emission scanning electron microscope (JSM-7001F, JEOL Ltd., Japan) was used to inspect the morphology of AOP and F-AOP samples. 2.4.5 Particle size and size distribution analysis The average particle size and zeta-potential of AOP or F-AOP in solutions (1.0, 2.0, 10.0 and 20.0 mg/mL) were determined using a Nicomp 380/ZLS Zeta potential/Particle sizer (PSS Nicomp, Santa Barbara, California, USA). All measurements were carried out at 25 °C. 2.5 In vitro antioxidant activities 2.5.1 DPPH radical scavenging activities The DPPH radical scavenging capacity of AOP and F-AOP were investigated according the previously published method, with slight modification 7


26

. Briefly,


polysaccharides were precisely weighed and dissolved in ultrapure water with various concentrations (0.1, 0.2, 0.5, 1.0, 2.0, 5.0, 10.0 mg/mL). Then, 2 mL of a freshly prepared 0.2 mM DPPH solution in ethanol was added to 2.0 mL of the AOP or F-AOP solution. The mixture was shaken and incubated at room temperature for 30 min in the dark, and the absorbance values were measured at 517 nm. The DPPH scavenging effect was calculated by the following formula: Scavenging rate (%) = (1 –

୅ଶି୅ଵ ୅଴

)×100

Here, A0 was the absorbance of the mixture with sample replaced by ultrapure water, A1 was the absorbance of the mixture with sample replaced by dehydrated alcohol, and A2 was the absorbance of the mixture of test sample solution. 2.5.2 Hydroxyl radical scavenging activities Scavenging hydroxyl radicals of AOP and F-AOP were investigated following the modified method 26. Concisely, polysaccharides were dissolved in ultrapure water with various concentrations (0.1, 0.2, 0.5, 1.0, 2.0, 5.0, 10.0 mg/mL). 1.0 mL of sample solution was homogeneous mixed with 1.0 mL of 2 mM FeSO4 and 1.0 mL of 6 mM salicylic acid alcohol solution. Then, after a second vortex, 1.0 mL 1 mM H2O2 was added. The mixture was kept at 37 °C for 60 min, then read absorbance at 510 nm. The hydroxyl radical scavenging activities were calculated following the equation below: Scavenging rate (%) = (1 –

୅ଵି୅ଶ ୅଴

)×100

Here, A0 was the absorbance of the mixture without sample solution; A1 was the absorbance of the mixture with sample; A2 was the absorbance of the mixture with ultrapure water instead of sample and H2O2. 2.5.3 Superoxide anion radical scavenging activities Superoxide anion radical scavenging activities of AOP and F-AOP were studied 8


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by the modified method

26

. Polysaccharides were dissolved in ultrapure water to

obtain various concentrations (0.1, 0.2, 0.5, 1.0, 2.0, 5.0, 10.0 mg/mL). Then, 0.35 mL sample solution was mixed with 5.0 mL 50 mM Tris-HCl buffer (pH 8.2). The mixture was cultured in water bath at 25 °C for 20 min, and then 0.2 mL 45 mM 1, 2, 3-phentriol were immediately added into the mixture and shaken rapidly. The change rate of absorbance was measured at 325nm every 30 s for 5 min. The superoxide anion radical scavenging activities were calculated following the equation below: Scavenging rate (%) =

୅଴ି୅ ୅଴

×100%

Here, A0 was the change rate of the control group; A was the change rate of the polysaccharide solution. 2.6 In vivo immunoregulatory activities 2.6.1 Animal treatment and experiment design Eighty female specific pathogen-free (SPF) BALB/c mice (8 weeks old, 20 ± 2 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd (Beijing, China, Certificate number: SCXK (Jing) 2016-0011). The mice were allowed to acclimatize the controlled conditions (25±1 °C; 40-50% relative humidity; 12 h light/dark cycle; free access to food and water) for 10 days. All experimental procedures involving animals and their care were approved by Nanchang University Medical College Animal Care Review Committee. The mice were randomized to eight groups (10 animals per group): (1) normal control group (NC), (2) model control CTX group (CTX), (3) low dose of AOP group with 50 mg/Kg/d, BW (AOP-L), (4) middle dose of AOP group with 100 mg/Kg/d, BW (AOP-M), (5) high dose of AOP group with 200 mg/Kg/d, BW (AOP-H), (6) low dose of F-AOP group with 50 mg/Kg/d, BW (FAOP-L), (7) middle dose of F-AOP group with 100 mg/Kg/d, BW (FAOP-M), (8) high dose of F-AOP group with 200 mg/Kg/d, BW (FAOP-H). The 9



administration concentration was referred to recommended allowances of the Pharmacopoeia of the People's Republic of China (Edition 2015). And the concentration of F-AOP was designed as an equivalent dosage of AOP. While NC group was intraperitoneally injected with saline, the other groups were intraperitoneally injected with 100 mg/Kg/d cyclophosphamide (CTX) for 3 days. In the week after modeling, all groups were intragastric administration with corresponding dose. 2.6.2 Detection of antibodies and cytokines in serum The levels of IgG and IgA in serum were detected with ThermoFisher Assay Kits (Fair Lawn, NJ, USA). The concentrations of interleukin (IL)-10 and interferon (IFN)-γ were detected with ELISA Kits (BOSTER Biological Technology Co., China). All procedures were performed according to the manufacturer’s instruction. 2.6.3 Relative expressions of splenic cytokines and transcription factors The levels of cytokines IL-10, interferon gamma (IFN-γ) and transcription factors T-box transcription factor (T-bet), GATA-binding protein-3 (GATA3), retinoid-related orphan receptor gamma t (RORγt), and forkhead box protein 3 (Foxp3) in spleen were detected with quantitative RT-PCR. The cDNA was prepared using high capacity cDNA reverse transcriptase kit. The qPCR was carried out on cDNA samples using the SYBR Green system (TaKaRa). Primers used were listed in Table 1. Data analysis was carried out on QuantStudioTM Real-Time PCR Software Version 1.1. The relative quantification value is expressed as 2-△△Ct, where △Ct is the difference between the mean Ct value of the sample and of the GAPDH control. 2.7 Statistical analysis GraphPad Prism® Software, version 7.0 was used for the calculation of 10


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statistical measures. Data are expressed as the mean ± SD. Statistical analysis was evaluated by one-way ANOVA with Tukey’s multiple tests. A p-value of < 0.05 or less was considered to be statistically significant. 3. Results and discussion

3.1 Characterization of AOP and F-AOP 3.1.1 Monosaccharide composition According to HPAEC analysis in Table 2, AOP was consist of rhamnose (Rha), arabinose (Ara), galactose (Gal), xylose (Xyl) and galacturonic acid (GalUA), with a molar ratio of 0.05 : 1 : 1.35 : 0.54 : 2.10. F-AOP showed a composition of Rha, Ara, Gal, GalUA and glucuronic acid (GlcUA), at a molar ratio of 0.07 : 1: 2.18 : 5.19 : 0.14. LAB fermentation significantly increased the proportions of both Rha and GalUA (p < 0.05), and decreased the contents of Xyl (p < 0.05). NCU116 fermentation made GlcUA detectable and made Xyl non-detectable. Study has confirmed that LAB fermentation process could change the monosaccharide composition

27

. In this present study, we observed that the monosaccharide

composition fermented with NCU116 was changed, as well as the elevated uronic acid content (GalUA and GlcUA). Generally, the monosaccharide compositions, molecular weight and other features were relative to the immunostimulatory activity of polysaccharides

13

. The alterations of immune activity between AOP and F-AOP

were further investigated. 3.1.2 Molecular weight As revealed in HPSEC chromatograms (Figure 1), both the two polysaccharides had a large range of molecular weights distribution. Here, the average molecular weights of AOP and F-AOP were 152.8 and 181.3 kDa, respectively. The molecular weight affected the behavior of polysaccharide. The decreased molecular weight of 11



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polysaccharide isolated from rice bran underwent Grifola frondosa fermentation, have proved to have an enhanced antioxidant and immune stimulation

27

. HPSEC

chromatograms of AOP and F-AOP presented less symmetrical peaks, suggesting that further purification is necessary to elucidate molecular structure of AOP and F-AOP. 3.1.3 Rheological behavior The rheological properties of AOP and F-AOP were investigated and presented in Figure 2. The shear viscosity was measured at 25 °C with shear rates in a range of 50 - 1000 s−1. The apparent viscosities of AOP and F-AOP solutions were at level of mPa. An elevated apparent viscosity was positively correlated with the increased concentrations of AOP and F-AOP. The increased viscosity at high concentration might be related to restrict polymer chain's movement and stretching, as a result of the corresponding increase in entanglements of individual chains

28

. Meanwhile, F-AOP

solutions displayed higher apparent viscosity than AOP solutions. This variation in viscosity might be attributed to different polysaccharides constructions between AOP and F-AOP, such as branched structure, molecular weight, and monosaccharide composition

29

. Linkage geometry of AOP and F-AOP remains to be determined in

future study. 3.1.4 Scanning electron micrographs analysis Surface morphology of AOP and F-AOP were measured by Scanning electron micrographs (SEM). The visible images of AOP and F-AOP exhibited quite similar surface, both with a thin slice shape (Figure 3). The different extractions and purification procedures for polysaccharide components have proved to be mainly responsible for the morphology changes 30. Considering that the extraction procedure of F-AOP was basically identical to AOP, we deduced that fermentation to raw materials had no visible alteration on surface morphology of its polysaccharide 12


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components. 3.1.5 Particle size and Zeta-potential values The particle sizes and Zeta-potential (Zp) values of AOP and F-AOP solution at different concentrations were listed in Table 3. The particle sizes of both AOP and F-AOP solutions were concentration-dependently increased. The droplet sizes of polysaccharide solutions generally increased with the increased concentrations

31

.

Here, the average particle size of F-AOP solution at 20 mg/mL were comparable to that of AOP solution at 5 mg/mL, indicating a considerable and significant difference on Zp values between F-AOP and AOP. Smaller droplets was linked to more stable nano-emulsions, and improved the bioavailability of natural products

32

, which

suggested F-AOP thus might possess superior bioavailability compared with AOP. Zp is an indicator of stabilities of polysaccharide solution. Generally, the absolute Zp value of a well stabilized solution was greater than |±30|

33

. At the low

concentration of 1 mg/mL, the Zp values of both AOP and F-AOP were over | ± 30 |, suggesting a good stability of both polysaccharide solution. However, the Zp values of other concentration were not close enough to | ± 30 |, and negatively correlated with the increased concentration. Thus, the polysaccharide concentration represents one of most influencing factors on Zp values for both AOP and FAOP samples, which is consistent with published study 34. 3.2 In vitro antioxidant activities 3.2.1 Scavenging effects on DPPH radicals Within the tested concentration range (from 0.05 mg/mL to 10 mg/mL), the scavenging abilities of AOP and F-AOP increased in a concentration-dependent manner (Figure 4A). F-AOP had a stronger DPPH free radical scavenging activity than that of AOP. Lower molecular weight polysaccharide (F-AOP) showed stronger 13



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DPPH radical scavenging capacity, because the low molecular-weight products had more access to free radicals, due to their stronger water-solubility and bigger surface area than high molecular weight polysaccharide (AOP) 35. 3.2.2 Scavenging effects on hydroxyl radicals The hydroxyl radical could easily react with biomacromolecules in biological tissues which contributed to cell damage

35

. The hydroxyl radical scavenging

activities of AOP and F-AOP revealed a concentration-dependent manner (Figure 4B). Consistently, the hydroxyl radical scavenging activity of F-AOP was stronger than that of equivalent AOP. The contents of uronic acid and protein, rather than neutral sugar contents determined the antioxidant activity of polysaccharide

17

. F-AOP with

higher uronic acid displayed stronger hydroxyl radical scavenging activity than AOP. 3.2.3 Scavenging effects on superoxide anion radicals Superoxide anion scavenging capacities of AOP and F-AOP were positively increased with the concentration variation (Figure 4C). F-AOP possessed relatively better superoxide anion scavenging activity than AOP, which can partially be attributed to their difference in molecular weight. Polysaccharide with different molecular weight distributions demonstrated different antioxidant capacities 27. Other structure features have also been demonstrated to influenced the antioxidant activities of polysaccharides, such as monosaccharide compositions, linkage patterns, and branching characteristics

14, 36

. Particularly, polysaccharide with higher content of

uronic acid, possessed a higher antioxidant capacity

35

, which are also supported by

the observed higher uronic acid content in F-AOP. 3.3 In vivo immunoregulatory activities 3.3.1 Effects of AOP and F-AOP on serum antibodies and cytokines levels The immunoregulatory activities of F-AOP and AOP were evaluated in CTX 14


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induced immunosuppressive mice models. CTX is a commonly cytotoxic drug that can suppress both humoral and cellular immunity. As shown in Figure 5, F-AOP (200 mg/Kg/d) significantly increased the contents of serum IgA and IgG levels, as well as AOP (200 mg/Kg/d). In addition, F-AOP (100 and 200 mg/Kg/d) significantly increased the level of IFN-γ and decreased the level of IL-10 in serum, as well as AOP (200 mg/Kg/d). T helper (Th) 1 cells make IFN-γ as their signature cytokine and IL-10 plays a critical role in the control of immune responses

37

. Therefore, F-AOP

and AOP at 200 mg/Kg/d displayed a coincident stimulation on the humoral immunity suppressed by CTX treatment. F-AOP particularly enhanced Th1 cellular immunity than the equivalent AOP. 3.3.2 Effects of AOP and F-AOP on splenic cytokines expression and T cell differentiation Th subsets have unique roles in mediating immune protection. For example, Th1 cells are responsible for cell-mediated immune responses, correspondingly Th2 cells are responsible for humoral-mediated immunity 37. The spleen with a highly organized lymphoid compartment, represents the most important immune organ for the combination of humoral and cellular immune response

38

. Focusing on distribution

and function of T cell subsets, the mRNA expression levels of special cytokines (IFN-γ, IL-17A, TGF-β and IL-10) and key nuclear transcription factor (T-bet, GATA-3, RORγt, STAT5 and Foxp3) were quantified by RT-qPCR (Figures 6 and 7). F-AOP (100 and 200 mg/Kg/d) significantly upregulated the expressions of splenic IFN-γ and T-bet compared with CTX treatment, as well as AOP (200 mg/Kg/d). Particularly, F-AOP at 50 mg/Kg/d significantly upregulated the level of T-bet compared with CTX group. Variation tendencies in the expressions of splenic T-bet and IFN-γ were almost consistent with the elevated secretion of serum IFN-γ, 15



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suggesting a promoted Th1 immune response driven by F-AOP. Moreover, AOP and F-AOP at 200mg/Kg/d significantly up-regulated GATA3 in spleen and up-regulated IgA and IgG in serum, demonstrating an elevated humoral defensive response. In addition, CTX group highly inhibited the expression of IL-10 in spleen compared with NC group, derived from its direct cytotoxic and depletion on regulatory T (Treg) cells 39. Therefore, the elevated content of serum IL-10 is mainly from the unexpected induction of myeloid-derived suppressor cells, a neglected aspect of CTX-mediated ablation of Treg cells 40. Both F-AOP and AOP minimally influence the expression of splenic Foxp3 (Fig.7E), as well as the expression of Treg associated IL-10 and TGF-β inhibited by CTX. TGF-β was required for the generation of Tregs by inducing the expression of Foxp3, which converted naive T cells differentiate into Tregs

41

. Thus, our results basically demonstrated similar variations between

Treg-specific Foxp3 and associated cytokines (IL-10 and TGF-β) in spleen. Th17 cells are a major source of IL-17 and IL-17 production maintaining a severe pro-inflammatory environment, responsible for excessive tissue damage

41

.

CTX has been demonstrated to induce CD4+ T cells expressing RORγt to become IL-17 producers in the spleen, with intestinal microbiota translocation being indispensable for this process 42. In signal pathways, STAT-stimulating cytokines are indispensable for the differentiation of inflammatory Th17 cells and drive IL-17 production41, 42. In the present study, all dosage of F-AOP and high dosage of AOP significantly decreased the expressions of splenic RORγt, STAT3 and IL-17, indicating an inhibition on CTX induced Th17 immune response. To systematically reflect the immunomodulation activities of polysaccharides administration, the mRNA expression ratio of T-bet/GATA-3 and RORγt/Foxp3 were assessed. F-AOP administration dose-dependently induced a gradual shift from Th17-dominant acute 16


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inflammatory response (expressions of IL-17 and RORγt) to Th1-dominant defensive immune response (expressions of IFN-γ and T-bet). Taken together, NCU116 fermentation influenced on the monosaccharide compositions, molecular weight, apparent viscosity, particle sizes, Zp values and SEM, antioxidant and immunoregulatory activities of polysaccharide isolated from asparagus. After fermentation, F-AOP increased the proportions of both Rha and GalUA, emerging GlcUA in F-AOP, which was not detectable in AOP. Fermentation decreased the average molecular weight from 181.3 kDa (AOP) to 152.8 kDa (F-AOP). The fermentation reduced the particle size and changed the rheology property. In vitro, NCU116 fermentation of asparagus pulps enhanced the DPPH, hydroxyl radical and superoxide anion radical scavenging abilities. In vivo, high dosages of AOP and F-AOP (200 mg/Kg/d) increased serum antibodies, maintaining Th2 subset. Particularly, F-AOP administration stimulated a gradual shift from Th17-dominant acute inflammatory response to Th1-dominant defensive immune response in a dose dependent manner. These results indicated that the NCU116 fermentation of pulps was practical and useful for obtaining functional polysaccharides with enhanced bioactivity. Conflicts of interest

The authors declare no conflicts of interest. Acknowledgments

This study was supported by the National Key Research and Development Program of China (2017YFD0400705-2), Jiangxi Provincial Major Program of Research and Development Foundation (Agriculture field, 20165ABC28004), Outstanding Science and Technology Innovation Team Project in Jiangxi Province (20165BCB19001), Collaborative Project in Agriculture and Food Field between 17



China and Canada (2017ZJGH0102001) and the Research Project of State Key Laboratory of Food Science and Technology (SKLF-ZZA-201611). 4. References

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10. Liu, G.; Sun, J.; He, X.; Tang, Y.; Li, J.; Ling, D.; Li, C.; Li, L.; Zheng, F.; Sheng, J., Fermentation process optimization and chemical constituent analysis on longan (Dimocarpus longan Lour.) wine. Food Chem. 2018, 256, 268-279. 11. Shi, J. J.; Zhang, J. G.; Sun, Y. H.; Qu, J.; Li, L.; Prasad, C.; Wei, Z. J., Physicochemical properties and antioxidant activities of polysaccharides sequentially extracted from peony seed dreg. Int. J. Biol. Macromol. 2016, 91, 23-30. 12. Chen, Y.; Zhang, H.; Wang, Y.; Nie, S.; Li, C.; Xie, M., Sulfated modification of the polysaccharides from Ganoderma atrum and their antioxidant and immunomodulating activities. Food Chem. 2015, 186, 231-238. 13. Ferreira, S. S.; Passos, C. P.; Madureira, P.; Vilanova, M.; Coimbra, M. A., Structure-function relationships of immunostimulatory polysaccharides: A review. Carbohydr. Polym. 2015, 132, 378-396. 14. Xu, Y.; Niu, X.; Liu, N.; Gao, Y.; Wang, L.; Xu, G.; Li, X.; Yang, Y., Characterization,

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Guan, J.; Zhang, D.; Lin, Y., Molecular Modification of Polysaccharides and Resulting Bioactivities. Compr. Rev. Food Sci. Food Saf. 2016, 15, 237-250. 16. Karaki, N.; Aljawish, A.; Humeau, C.; Muniglia, L.; Jasniewski, J., Enzymatic modification of polysaccharides: Mechanisms, properties, and potential applications: A review. Enzyme Microb. Technol. 2016, 90, 1-18. 17. Wang, Y.; Shao, S.; Xu, P.; Chen, H.; Linshiau, S. Y.; Deng, Y. T.; Lin, J. K., Fermentation process enhanced production and bioactivities of oolong tea polysaccharides. Food Res. Int. 2012, 46, 158-166. 18. Jegou, S.; Hoang, D. A.; Salmon, T.; Williams, P.; Oluwa, S.; Vrigneau, C.; Doco, T.; Marchal, R., Effect of grape juice press fractioning on polysaccharide and oligosaccharide compositions o Pinot meunier and Chardonnay Champagne base wines. Food Chem. 2017, 232, 49-59. 19. Feller, C.; Richter, E.; Smolders, T.; Wichura, A., Phenological growth stages of edible asparagus (Asparagus officinalis): codification and description according to the BBCH scale. Ann. Appl. Biol. 2012, 160, 174-180. 20. Benson, B., 2009 update of the world's asparagus production areas, spear utilization and production periods. Acta Hortic. 2012, 950, 87-100. 21. Kou, X.; Mao, C.; Xie, B.; Li, X.; Xue, Z.; Zhang, Z., Functional characterization of oligosaccharides purified from Asparagus officinalis peel. J. Food Nutr. Res.

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Tikkanen-Kaukanen, C., Emulsification properties of polysaccharides from Dioscorea opposita Thunb. Food chem. 2017, 221, 919-925. 32. Gómez-Mascaraque, electrosprayed

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mechanisms of T cell modulation. Cancer Treat. Rev. 2016, 42, 3-9. 40. Becker,

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41. Raphael, I.; Nalawade, S.; Eagar, T. N.; Forsthuber, T. G., T cell subsets and their signature cytokines in autoimmune and inflammatory diseases. Cytokine. 2015, 74, 5-17. 42. Daillère, R.; Vétizou, M.; Waldschmitt, N.; Yamazaki, T.; Isnard, C.; Poirier-Colame, V.; Duong, C. P.; Flament, C.; Lepage, P.; Roberti, M. P., Enterococcus

hirae

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cyclophosphamide-induced therapeutic immunomodulatory effects. Immunity. 2016, 45, 931-943. Figure captions Figure 1. HPSEC profiles of AOP and F-AOP. (A) AOP; (B) F-AOP. Figure 2. The apparent viscosity of AOP and F-AOP at different concentrations for

the shear rates in the range of 50-1000 s-1. Both AOP and F-AOP were dissolved in ultrapure water with various concentrations of 5 mg/mL, 10 mg/mL and 20 mg/mL. Figure 3 Scanning electron micrographs of AOP and F-AOP at different

magnifications. (A) AOP at magnification of ×250; (B) F-AOP at magnification of ×250; (C) AOP at magnification of ×10000; (D) F-AOP at magnification of ×10000. Figure 4. Antioxidant activities of AOP and F-AOP in vitro. (A) DPPH radical

scavenging activities of AOP and F-AOP; (B) hydroxyl radical scavenging activities of AOP and F-AOP; (C) superoxide anion radical scavenging activities of AOP and F-AOP. Figure 5. Effects of AOP and F-AOP on serum antibodies and cytokines levels. (A) 23



the level of IgA in serum; (B) the level of IgG in serum; (C) the level of IFN-γ in serum; (D) the level of IL-10 in serum. NC for blank group treated only with saline solution (0.9%, w/v); CTX for cyclophosphamide group treated with CTX (100 mg/kg ); AOP-L for AOP-L group treated with CTX (100 mg/kg) and AOP 50 mg/kg; AOP-M for AOP-M group treated with CTX (100 mg/kg) and AOP 100 mg/kg; AOP-H for AOP-H group treated with CTX (100 mg/kg) and AOP 200 mg/kg; FAOP-L for FAOP-L group treated with CTX (100 mg/kg) and F-AOP 50 mg/kg; FAOP-M for FAOP-M group treated with CTX (100 mg/kg) and F-AOP 100 mg/kg; FAOP-H for FAOP-H group treated with CTX (100 mg/kg) and F-AOP 200 mg/kg; Values were expressed as the mean ± SD (n = 9). Significance difference were *p < 0.05, **p < 0.01 as compared with CTX group. Figure 6. Effects of AOP and F-AOP on splenic cytokines expression. The relative

levels of IFN-γ (A); IL-17 (B); and IL-10 (C). Values were expressed as the mean ± SD (n = 6). Significance difference were *p < 0.05, **p < 0.01 as compared with CTX group. Figure 7. Effects of AOP and F-AOP on splenic T cell differentiation. The mRNA

relative expressions of T-bet (A); GATA3 (B); RORγt (C); STAT3 (D); Foxp3 (E); TGF-β (F). Values were expressed as the mean ± SD (n = 6). Significance difference were *p < 0.05, **p < 0.01 as compared with CTX group. Figure 8. Effect of fermentation on the mRNA expression ratio of T-bet/GATA-3 and

RORγt/Foxp3. The mRNA expression ratio of T-bet/GATA-3(A); and RORγt/Foxp3 (B). Values were expressed as the mean ± SD (n = 6). Significance difference were *p < 0.05, **p < 0.01 as compared with CTX group.

24


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Table 1. The primer sequences for qRT-PCR (5’→3’)

Forward primer

Reverse primer

IL-10

5’-TGTCAAATTCATTCATGGCCT-3’

5’-ATCGATTTCTCCCCTGTGAA-3’

IL-17

5’-TGAGCTTCCCAGATCACAGA-3’

5’-TCCAGAAGGCCCTCAGACTA-3’

IFN-γ

5’-TGAGCTCATTGAATGCTTGG-3’

5’-ACAGCAAGGCGAAAAAGGAT-3’

T-bet

5’-CTGGAGCCCACAAGCCATTA-3’

5’-TTTCCACACTGCACCCACT-3’

GATA3

5’-AGGATGTCCCTGCTCTCCTT-3’

5’-GCCTGCGGACTCTACCATAA-3’

RORγt

5’-GCAGGGCCTACAATGCCAAC-3’

5’-GAACCAGGGCCGTGTAGAGG-3’

STAT3

5’-AACGACCTGCAGCAATACCA-3’

5’-TCCATGTCAAACGTGAGCGA-3’

Foxp3

5’-CTCGTCTGAAGGCAGAGTCA-3’

5’-TGGCAGAGAGGTATTGAGG-3’

TGF-β

5’-CTGGATACCAACTACTGCTTCAG-3’

5’-TTGGTTGTAGAGGGCAAGGACCT-3’

GAPDH

5’-AGGTCGGTGTGAACGGATTTG-3’

5’-TGTAGACCATGTAGTTGAGGTCA-3’

25



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Table 2. Effect of fermentation on the monosaccharide composition and molecular

weight. Sample

AOP

F-AOP

Rhamnose

7.61±1.11a

54.31±2.85b

Arabinose

137.27±18.20a

70.91±2.23b

Galactose

221.76±21.84a

185.53±3.82b

Glucose

ND

ND

Xylose

74.61±13.11a

NDb

Galacturonic acid

361.94±35.34a

476.03±17.14b

Glucuronic acid

NDa

12.75±0.67b

181.3a

152.8b

Monosaccharide composition (‰)

Molecular weight (kDa)

Values are expressed as means ± SD and three replicated independent determinations; ND, non-detectable or lower than the limit of quantification. Values with different letters in the same line indicate statistically significant differences (p < 0.05).

26


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Table 3. Particle sizes and Zeta-potential of AOP and F-AOP at different

concentrations. Sample

AOP

F-AOP

1 mg/mL

1.23±0.05a

0.58±0.03b

5 mg/mL

1.83±0.03a

0.77±0.02b

10 mg/mL

2.67±0.11a

0.97±0.03b

20 mg/mL

3.63±0.24a

1.64±0.07b

1 mg/mL

-32.15±0.45a

-31.5±0.30a

5 mg/mL

-21.2±0.91a

-21.4±1.27a

10 mg/mL

-18.43±0.80a

-19.68±2.07a

20 mg/mL

-16.55±0.81a

-18.40±1.30a

Particle size

Zeta-potential values

Values are expressed as means ± SD and three replicated independent determinations. Values with different letters in the same line indicate statistically significant differences (p < 0.05).

27



Figure 1

28


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Figure 2

29



Figure 3

30


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Figure 4

31



Figure 5

32


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Figure 6

33



Figure 7

34


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Figure 8

35



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Graphic for manuscript In this work, a selected lactic acid bacteria strain NCU116 was used to ferment Asparagus officinalis pulps. NCU116 fermentation changed the physicochemical characterizations of Asparagus officinalis polysaccharide (AOP). In vitro, fermentedpolysaccharide displayed superior free radical scavenging properties. In vivo, fermented-polysaccharide exhibited enhanced Th1 immune response. Asparagus officinalis L. LAB fermentation

Monosaccharide compositions

Polysaccharides

AOP and F-AOP

extraction

Molecular weight

Physicochemical characterizations

Apparent viscosity Solution stability

Fermented asparagus pulps

Bioactivity evaluation In vitro antioxidant activities • DPPH scavenging ability • Hydroxyl radical scavenging ability • Superoxide anion radical scavenging ability.

In vivo immunoregulatory activities • Serum antibodies: • Th1 • Th2 • Th17


Foxp3

T-bet

Treg

Th1

F-AOP GATA3 Th2

RORγt Th17

Effect of Lactobacillus plantarum NCU116 Fermentation on Asparagus

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