Journal of Food Science and Technology

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Journal of Food Science and Technology Production, partial characterization and antioxidant properties of exopolysaccharide αD-glucan produced by Leuconostoc lactis KC117496 isolated from an idli batter --Manuscript Draft-Manuscript Number:

JFST-D-18-01252R1

Full Title:

Production, partial characterization and antioxidant properties of exopolysaccharide αD-glucan produced by Leuconostoc lactis KC117496 isolated from an idli batter

Article Type:

Original Article

Corresponding Author:

Prathap Kumar Shetty, Ph.D Food Science and TechnologyPondicherry University INDIA

Order of Authors:

Chinnashanmugam Saravanan, Ph.D Digambar Kavitake, Research Scholar Sujatha Kandasamy, Ph.D Palanisamy Bruntha Devi, Ph.D Prathapkumar Halady Shetty, Ph.D

Corresponding Author Secondary Information: Corresponding Author's Institution:

Food Science and TechnologyPondicherry University

Corresponding Author's Secondary Institution: First Author:

Chinnashanmugam Saravanan, Ph.D

First Author Secondary Information: Order of Authors Secondary Information: Funding Information: Abstract:

Exopolysaccharide (EPS) produced from Leuconostoc lactis KC117496 isolated from the naturally fermented idli batter has been reported earlier. Here, study aimed to optimize the carbon source to enhance the yield of EPS production, partial characterization and antioxidant activity of α-D-glucan EPS. Among different disaccharides (sucrose, maltose, lactose) and monosaccharides (glucose, galactose and fructose), combination of sucrose and glucose at 2 % showed highest EPS production of 4.55 g/L in MRS medium. The molecular weight was identified as ~4.428 x 103 kDa with MALDI-TOF mass spectroscopy. 1D and 2D NMR results exhibited the presence of α-1-6 and α-1-3 linked glucose revealed EPS as a glucan. Antioxidant properties of glucan (500 µg/mL) revealed the significant oxidation alleviation potential such as DPPH (74 %) and Hydroxyl radical activity (97.8 %), whereas metal chelating activity (70 %) was lower as compare to control standard. These characteristics of glucan EPS reveals its potential application in food and pharmaceutical industry.

Suggested Reviewers:

Dr. Vijay K. Juneja, Ph.D [email protected] Dr. V. Ravishankar Rai [email protected] Cherl-Ho Lee, PhD Professor, Korea University, Seoul South Korea [email protected]

Response to Reviewers:

Response to the reviewer's comments has been attached as a separate file.

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Authors' Response to Reviewers' Comments

Click here to access/download;Authors' Response to Reviewers' Comments;Response to reviewers comments.docx

Response to Reviewer’s Comments Authors would like to express our gratitude to all the reviewers for their careful reading of the manuscript and constructive comments. As per the reviewer’s comments and suggestions, we have modified and improved the current manuscript. Response to reviewer’s comment has been given as mentioned below and highlighted in the manuscript.

Reviewer #1: The aim of manuscript is to analyze production, characterization and antioxidant properties of exopolysaccharide α-D-glucan produced by Leuconostoc lactis KC117496 isolated from an idli batter. It deals with interesting issue, but novelty and scientific importance of study not described well in introduction. Authors should consider some suggestion for further improvement of manuscript: Necessary to add few sentences and describe idli batter for international readers L 51 was it naturally fermented or commercial one? Naturally fermented. L 52-57 Add models and producers of used equipment. How optical density was assessed (equipment, preparation of samples and etc) Text has been modified as per the suggestion. L 53 Necessary to add details about L.lactis Details of L.lactis is already added in the introduction part. L 123 Was excel used for calculation. Correction of data analysis description is necessary necessary to use SI units thorough manuscript Yes, Excel has been used. All the units has been changed to SI unit system. Figure 2 and figure 6 necessary to add letters to show if significant differences were found depending on analysed factors. more wide discussions and comparison of study results with similar data is necessary. Results and figures are revised. Statistical data analysis and interpretation is missing Statistical analysis has been added and interpretations added.

Reviewer #2: The manuscript entitled „Production, characterization and antioxidant properties of exopolysaccharide α-D-glucan produced by Leuconostoc lactis KC117496 isolated from an idli batter" requires a significant correction. Minor errors: *

Highlights 1 - should be up to 125 characters (now is 127). Highlights 1 has been revised.

* Fig. 2 - the description does not correspond with the graph. What are the parts C and D on the chart? The quality of the drawings (letters A, B, C, D) needs improvement. Description and figure 2 has been revised as per the suggestions. *

References to Fig. 3 and Fig. 4 are not included in the text. Fig. 3 and Fig. 4 are added in the text

*

Fig. 5 should be removed.

Fig. 5 is a representation of the MALDI-TOF-MS analysis and hence removal may lead to loss of clarity. Reviewer #3: Comments - further recommendations This study aimed to study and characterize the antioxidant properties of α-D-glucan EPS. So, this paper may be accepted for publication after effectuating minor revisions. 1. Abstract: - The authors should re-arrange and re-write the abstract as the following order (Problem of research, aim of study, remarkable methodology, remarkable results, and significance of study). Abstract has been revised accordingly. 2. Conclusions: - This section need to be improved. Conclusion part has been revised and improved further.

Reviewer #4: Abstract: Okay, but does not well capture the justification and significance of the study. Abstract has been revised.

Line 8-9: Recast statement for clarity of result. Statements recasted accordingly. Introduction Line 16-17: Recast as benefits such as increased digestibility, enhanced flavour, improved nutritional and pharmacological values. Recasted as per the suggestions. Line 22: homopolysaccharide that are produced Sentence has been corrected Line 36: what is the meaning of Btq? Btq is a EPS produced by Bacillus tequilensis. Reference for the same has been cited in the text. Line 39: Change through to by Changed as per the suggestion. Line 42-44: Furthermore, EPS has been intensively employed Changed as per the suggestion. Line 46: In present study, α-D-glucan EPS was partially characterization and the antioxidant properties determined Changed as per the suggestion. Line 55: Up to Corrected. Line 63-64: Please explain/describe briefly how the growth and EPS production was determined? Growth has been compared by optical density and the specific gravity analysis whereas EPS production has been determined by phenol-sulfuric acid method. Line 66: Which enzyme was inactivated? Describe briefly; Was it intracellular or extracellular? Heat treatment is done basically to inactivate the extracellular enzymes such as glycansucrases. Line 91-120: Please write all formula using Microsoft Equation Suggestion implied in the manuscript.

Results and Discussion: The significance and impact of the results are not adequately or extensively discussed. Results and Discussion has been modified as suggested. Line 136: What is the implication on EPS production? Here we have compared the doubling time of culture at different shaking conditions. The lesser the doubling time reveals the fast is growth of the bacteria. It is having direct relevance with growth, which is one of the parameter which effects EPS production. Line 179-181: What is the implication of this radical scavenging data; what impact will it have on health or a living organism Line 184: Same as above These antioxidant assays reveal the oxidation alleviation potential of this glucan EPS, which can protect the oxidative cell damage.

Reviewer #5: Manuscript JFST-D-18-01252, entitled "Production, characterization and antioxidant properties of exopolysaccharide α-Dglucan produced by Leuconostoc lactis KC117496 isolated from an idli batter" The 1st highlight is too long. Highlight has been revised. Abstract Ln 6: SI units should be used. The sentence is poorly constructed and not clear. SI unit has been added and sentence has been revised. Ln 8: "Confirming" is not clear. Please be more precise Sentence revised accordingly. Ln 10: "control" is completely not clear Control has been changed to control standard. Because there are different control standards for antioxidant assay and metal chelating activity. Introduction

Ln 28: SI units should be used. SI unit has been added. Ln 31-36: The unit mg/L must be explained in detail. Otherwise it is hard to follow. SI unit has been added. The novelty of the study should be better emphasized in introduction section. Novelty and significance has been mentioned in the abstract as well as conclusion part. Ln 36: "Btq" is not clear Btq is a EPS produced by Bacillus tequilensis. Reference for the same has been cited in the text. Ln 45: The scientific style should be used. Sentence has been revised as suggested. Ln 46-47: The aim of the work is not clear. Please be more precise. Aim of the work has been elaborated in the revised draft. Materials and methods Ln 53: "10 mL of L. lactis" is completely not informative. CFU/mL should be specified. Revised as per the suggestion. Ln 53-89: The references to the methods applied (also preparation methods) are missing/not clear. Who exactly is the Author of the methods applied? Are these methods valid? Without sufficient information it is hard to reproduce the results, follow, compare the idea and the obtained results with the literature. References are cited in the respective sections as suggested. Ln 68-69: What you mean by "treated" here? Please be more precise. Appropriate literature data should be also provided. Here Sevage reagent (chloroform: n-butanol at 5:1 v/v) was added to the supernatent to remove the proteinaceous materials. Ln 72: Improper unit notation. Unit has been corrected.

Ln 73-74: What happened with EPS solution after dialysis? The method should be clearly described. The old colorimetric method provides proximate results, often overestimated by the presence of other sugars/contaminants which interfere with phenol reagent. For precise measuring EPS yield, what is important in this research work I recommend to use modern technique as HPLC or GC for quantification of polysaccharides. In the EPS extraction process, Sevage reagent (chloroform: n-butanol at 5:1 v/v) has been used to remove the proteinaceous materials. Then chilled ethanol has been added for EPS precipitation (monosaccharides will not get precipitate in the ethanol). The dialysis is for the removal of unwanted small molecules such as salts, reducing agents from the larger macromolecules like EPS. We have used MALDI-TOF instead of HPLC (as per Wang, J., Kalt, W., & Sporns, P. 2000 where they studied comparison between HPLC and MALDI-TOF MS analysis of anthocyanins in highbush blueberries. Journal of Agricultural and Food Chemistry, 48(8), 33303335.) How the yields were determined exactly? This is not clearly written in M&M. Quantification of EPS was done using phenol sulphuric acid method (Dubois et al. 1956) Ln 48-89: The method is poorly described. What is the "sample" in ln 86? What is the reason of application of this method for EPS characterization? What is the advantage of this method compared to HPSEC which is commonly used for Mw analysis? How the system was calibrated? This all should be clearly written. The discussion provided in Ln 168-172 is poor and shallow. MwD profiles should be shown. On the basis of SEC profiles, it could be possible to discuss the effectiveness of purification steps applied. Methods are revised as per the suggestions. In line 86, the mentioned sample is of EPS (also corrected in the text). MALDI-TOF MS proved to be more rapid in the accurate identification and quantification of different masses. The whole M&M section needs substantial revision. General remark to the discussion - In my opinion, the discussion provided by Authors is not possible to follow and verify, because of missing very important details in the methodology section. Due to a poorly presented material and methods, I am not able to follow and properly review the discussion. Due to poorly presented material and methods the conclusions are for me confusing and not convincing. Materials and methods are substantially revised to improve the clarity.

Reviewer #6:

The study describes the characterization and bioactive potential of EPS produced by Leuconostoc lactis KC117496. The experimental have been well carried out and minor revisions have been suggested. 1. Microstructural studies for EPS can be performed. 2. IR studies for functional group determination. 3. Physicochemical characterization of EPS for solubility in water/organic solvents. 4. Determination of thermal stability and degradation temperature for food appliactions. 5. Determination of biomass during optimum EPS production. Above suggested studies has been reported in previous manuscript as mentioned in the text (Saravanan C, Shetty PKH (2016) Isolation and characterization of exopolysaccharide from Leuconostoc lactis KC117496 isolated from idli batter. Int J Biol Macromol 90: 100-106.).

Reviewer #9: Present manuscript describes the optimization of EPS production from Leuconostoc lactis KC117496 and its antioxidant activity. It is a follow up of earlier publication (Saravanan C, Shetty PKH .2016. Isolation and characterization of exopolysaccharide from Leuconostoc lactis KC117496 isolated from idli batter. Int J Biol Macromol 90: 100-106). Here, additional data of the EPS characterization (MALDI-TOF for molecular weight and additional NMR data) included. My concerns are 1. EPS production optimization experiments are not clear. Authors claimed that 2% sucrose as optimum concentration but nowhere mentioned about the results for the range of concentrations tried. EPS production at different concentrations of sucrose has been carried out and results has been added as a supplement 1. 2. Optimization focused more on RPM and time, what about other parameters? (Temperature, pH, nitrogen source, inoculum load, etc.). The optimization of EPS production is incomplete. Different bacteria having capacity to produce various types of EPS at different growth conditions. Shaking conditions (RPM) is one of the aspect playing major role in the EPS production. Bacteria produces EPS at different time durations, few utilizes these EPS again for their growth and survival. So it is important to study the incubation time and where

optimum yield of EPS can be achieved. Sucrose is an enhancer for EPS production, different concentration of sucrose effects significantly on EPS production, hence we studied here. 3. Most of the EPS characterization has been claimed already in the Int J Biol Macromol paper. Please focus on the EPS production optimization and antioxidant properties and remove the "characterization" from the title. As per the suggestion, we have changed the ‘characterization’ to ‘partial characterization’

Cover Letter

Cover letter From Dr. Prathapkumar H Shetty Associate Professor & Head Department of Food Science and Technology Pondicherry University Pondicherry, India.

To The Chief Editor

Journal of Food Science and Technology Dear Sir, Please

find

herewith

our

revised

manuscript

titled,

“Production,

partial

characterization and antioxidant properties of exopolysaccharide α-D-glucan produced by Leuconostoclactis KC117496 isolated from idli batter” for possible publication as an “Original Article” in your revered journal. Exopolysaccharide (EPS) produced from Leuconostoc lactis KC117496 isolated from the naturally fermented idli batter has been reported earlier. Here, study aimed to optimize the carbon source to enhance the yield of EPS production, partial characterization and antioxidant activity of α-D-glucan EPS. Among different disaccharides (sucrose, maltose, lactose) and monosaccharides (glucose, galactose and fructose), combination of sucrose and glucose at 2 % showed highest EPS production of 4.55 g/L in MRS medium. The molecular weight was identified as ~4.428 x 103 kDa with MALDI-TOF mass spectroscopy. 1D and 2D NMR results exhibited the presence of α-1-6 and α-1-3 linked glucose revealed EPS as a glucan. Antioxidant

properties of glucan (500 µg/mL) revealed the significant oxidation alleviation potential such as DPPH (74 %) and Hydroxyl radical activity (97.8 %), whereas metal chelating activity (70 %) was lower as compare to control standard. These characteristics of glucan EPS reveals its potential application in food and pharmaceutical industry. We are assuring that the work has not been published elsewhere; either completely or in partial format and the manuscript has not been submitted to any other journal. The manuscript has been drafted according to the prescribed format and guidelines. The present study did not include any animal or human studies. All the authors are concurring with the submission of the manuscript. Hope, you will find the manuscript in order. Kindly process it at the earliest time possible. Thanking you.

Yours sincerely, Prathapkumar H Shetty

Title Page

Production, partial characterization and antioxidant properties of exopolysaccharide αD-glucan produced by Leuconostoc lactis KC117496 isolated from an idli batter Chinnashanmugam Saravanan, Digambar Kavitake, Sujatha Kandasamy, Palanisamy Bruntha Devi, Prathapkumar Halady Shetty1 Department of Food Science and Technology, Pondicherry University, Pondicherry 605014, India

Highlights 

EPS yield of L. lactis KC117496 was optimized up to 4.55 g/L using sucrose and glucose as carbon sources in MRS media 



Structural investigation of EPS showed α-1→6 and α-1→3 linked glucose through NMR analysis



The molecular weight of glucan was identified as ~4.428 x 103 kDa



Antioxidant properties and metal chelating activity of glucan were studied

1

Corresponding author. Tel.: (+91-944.2293718)

E-mail address:[email protected] (Dr.Prathapkumar H Shetty)

1

Blinded Manuscript

Click here to view linked References

Production, partial characterization and antioxidant properties of exopolysaccharide α1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

D-glucan produced by Leuconostoc lactis KC117496 isolated from an idli batter Chinnashanmugam Saravanan, Digambar Kavitake, Sujatha Kandasamy, Palanisamy Bruntha Devi, Prathapkumar Halady Shetty1 Department of Food Science and Technology, Pondicherry University, Pondicherry 605014, India

Highlights 

EPS yield of L. lactis KC117496 was optimized up to 4.55 g/L using sucrose and glucose as carbon sources in MRS media 



Structural investigation of EPS showed α-1→6 and α-1→3 linked glucose through NMR analysis



The molecular weight of glucan was identified as ~4.428 x 103 kDa



Antioxidant properties and metal chelating activity of glucan were studied

1

Corresponding author. Tel.: (+91-944.2293718)

E-mail address:[email protected] (Dr.Prathapkumar H Shetty)

1

1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

Abstract

2

Exopolysaccharide (EPS) produced from Leuconostoc lactis KC117496 isolated from

3

the naturally fermented idli batter has been reported earlier. Here, study aimed to optimize the

4

carbon source to enhance the yield of EPS production, partial characterization and

5

antioxidant activity of α-D-glucan EPS. Among different disaccharides (sucrose, maltose,

6

lactose) and monosaccharides (glucose, galactose and fructose), combination of sucrose and

7

glucose at 2 % showed highest EPS production of 4.55 g/L in MRS medium. The molecular

8

weight was identified as ~4.428 x 103 kDa with MALDI-TOF mass spectroscopy. 1D and 2D

9

NMR results exhibited the presence of α-1-6 and α-1-3 linked glucose revealed EPS as a

10

glucan. Antioxidant properties of glucan (500 µg/mL) revealed the significant oxidation

11

alleviation potential such as DPPH (74 %) and Hydroxyl radical activity (97.8 %), whereas

12

metal chelating activity (70 %) was lower as compare to control standard. These

13

characteristics of glucan EPS reveals its potential application in food and pharmaceutical

14

industry.

15

Keywords: Exopolysaccharide, optimization, characterization, NMR, MALDI-TOF/TOF,

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Antioxidant activity.

2

17 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

Introduction

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Indian traditional fermented foods are rich source of Lactic Acid Bacteria (LAB)

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being widely consumed as a healthy diet with numerous benefits such as increased

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digestibility, enhanced flavour, improved nutritional and pharmacological values. Idli is one

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of the well-known, easily digestible, Indian traditional fermented food prepared from rice and

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black gram dhal. Most of the LAB involved in fermentation of idli batter are capable of

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producing exopolysaccharide (EPS) that are known to improve the texture and sensory

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properties of idli (Prajapati, 2013; Patel et al. 2014; Saravanan et al. 2015). Furthermore, the

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EPS has wide application as bio-flocculants, stabilizers, emulsifiers, bio-absorbents, drug

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delivery agents and heavy metal removing agents (Liu et al. 2010). Dextran is a

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homopolysaccharide which is produced by various LAB, especially Leuconostoc and

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Streptococcus sp. in sucrose containing media (Kim et al. 2003). Glucan EPS produced by

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Leuconostoc garlicum PR encompasses 95% α-1→6 glucopyranose linkage along with low

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content of α-1→2, α-1→3 and α-1→4 linkages (Capek et al. 2011). Leuconostoc

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pseudomesenteroides XG5 produced water soluble EPS composed of glucose subunits with

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α-(1-6) linkage (Dextran) and the molecular mass was 2.6 × 103 kDa (Zhou et al. 2017).

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Molecular weight of EPS produced by Leuconostoc citreum-BMS, L. mesenteroides-TMS,

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Pediococcus pentosaceus-DPS and L. pseudomesenteroides-CM were around 106 Da (Abid et

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al. 2017). Joshi and Koijam (2014) isolated L. lactis from ethnically fermented beverage

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(Kyiad pyrsi) that yields EPS at a maximum of 0.340 g/L. Under normal conditions, EPS

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produced from LAB ranged between 100-196 mg/L, while homopolysaccharides produced

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are less than 1g/L. EPS produced by L. lactis and L. mesenteroides isolated from raw milk

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ranges between 0.8–0.9 g/L (Van der Meulen et al. 2007). Under optimized conditions,

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glucan production by L. dextranicum NRRL B-1146 reached a maximum of 1.063 g/L

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(Majumder et al. 2009). EPS produced by Bacillus tequilensis FR9 showed potential strong

3

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antioxidant effects through scavenging reactive oxygen species (ROS) (Rani et al. 2017). The

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commercial glucan with different molecular weight (1, 4, and 14.7 × 104 Da) synthesized by

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L. mesenteroides exhibited strong antioxidant, anticoagulant and immunomodulatory

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activities (Soeiro et al. 2016). The in vitro antioxidant properties of the EPS showed

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remarkable scavenging effects on superoxide anion radical and hydroxyl radical (Adesulu-

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Dahunsi et al. 2018). Furthermore, EPS has been intensively employed as a food additive in

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texture improvement which impart the development of innovative food products with

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enhanced appearance, mouth feel, firmness and rheological properties (De Vuyst et al. 2001).

50

Previously, we have reported this EPS as α-D-glucan along with production of 0.360

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g/L from Leuconostoc lactis KC117496 isolated from fermented idli batter (Saravanan and

52

Shetty 2016). In the present study, optimization of carbon source, partial characterization and

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antioxidant property of α-D-glucan EPS has been studied to improve its production and

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bioactive potential.

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Materials and Methods

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Microbial culture and chemicals

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The EPS producing strain Leuconostoc lactis KC117496 used in this study was

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isolated from naturally fermented idli batter. All reagents used were of analytical grade.

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Growth condition of Leuconostoc lactis KC117496

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For EPS production, 10 mL of L. lactis grown in MRS broth for 24 h (1x106 cfu/mL)

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was used as an inoculum in 100 mL MRS broth supplemented with 2% sucrose. Flasks were

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kept for incubation at 30 °C under different shaking conditions (100, 125 and 150 rpm) up to

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48 h. At every 8 h intervals, optical density (O.D.) was assessed at 600 nm by using

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spectrophotometer (UV-1800, Shimadzu, North America) and growth curves were plotted.

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Extraction and purification of EPS

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At the time of harvesting, 48 h grown culture broth was subjected to 100 °C for 10

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min for enzyme inactivation by heating the cell suspension. After cooling to room 4

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

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temperature, the biomass was removed by centrifugation at 4100 xg for 20 min. The resulting

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supernatant was treated with 2 % Trichloroacetic acid to remove the proteinaceous materials

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by centrifugation. Then EPS was precipitated using thrice the volume of ice cold ethanol and

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left overnight. The precipitated EPS was separated via centrifugation (19200 xg, 15 min) and

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dissolved in Milli Q water. For further purification, EPS was encased in a dialysis bag (12-14

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kDa) and dialysed until 48 h at 4 °C in Milli Q water (Saravanan and Shetty 2016). Dialyzed

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EPS was subjected to lyophilization. Quantification of EPS was done using phenol sulphuric

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acid method (Dubois et al. 1956).

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Optimization kinetics of L. lactis KC117496 to increase EPS production

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The batch fermentation was tested initially for EPS production using different

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disaccharides such as sucrose, lactose and maltose as sole carbon source (2 % w/v).

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Subsequently, the disaccharide that yields the highest EPS was further combined with

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monosaccharide (2 % w/v) such as glucose, mannose and galactose after 8 h of incubation.

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Freshly prepared 10 % inoculum (24 h) of L. lactis was added into each flask. EPS

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production was observed at every 8 h interval up to 48 h under shaking conditions.

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Nuclear magnetic resonance spectroscopy analysis

84

In structural analysis, 1H and

13

C NMR spectrum was measured using the partially

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purified EPS (10 mg) dissolved in 99.96% D2O as per the method described by Liu et al.

86

(2007). 1H NMR spectra was carried out at 1.00 delay (D1) and 3.17 s acquisition time (AQ),

87

while for

88

spectroscopy (COSY),

89

(HSQC) and 1H-13C heteronuclear multiple bond correlation spectroscopy (HMBC) were also

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done by using Bruker DRX Advance 400 MHz spectrometer.

91

MALDI-TOF/TOF mass spectrometry

13

C NMR the D1 and AQ was 2.0 and 1.1 s, respectively. 1H-1H correlation 1

H-13C heteronuclear single quantum coherence spectroscopy

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Matrix-assisted laser desorption ionization (Ultraflex TOF/TOF) mass spectrometry

93

was executed in positive and negative modes, with an alpha-cyano-4-hydroxycinnamic acid 5

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

94

(10 mg/mL in 50% acetonitrile, 0.1% TFA) matrix (Ismail and Nampoothiri 2010). EPS (0.5

95

µl) was taken up with a nitrogen laser (k 330 nm) via a detector. Mass spectra were detailed

96

over a range of 0–16000 m/z and minimum 5000 laser shots were taken per spectrum with the

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laser repetition rate 2000 Hz.

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Antioxidant properties of EPS

99

2,2-diphenyl-1-picrylhydrazyl radicals scavenging activity

100

The assay was executed as per the method described by Yang et al. (2006) along with

101

slight modification. For reaction, 100 µl of 0.2 mM DPPH (95% ethanol v/v) was added to

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100 µl EPS solution at various concentrations (100-500 µg) and incubated in dark room for

103

30 min at room temperature. The absorbance (517 nm) was compared with control solution of

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DPPH without EPS and a positive control (Ascorbic acid). The scavenging activity % =

1 − 𝐴𝐶𝑜𝑛𝑡𝑟𝑜𝑙 − 𝐴𝑇𝑒𝑠𝑡 𝐴𝐶𝑜𝑛𝑡𝑟𝑜𝑙

× 100

105 106

whereas, AControl is the absorbance with only DPPH solution, ATest is the absorbance

107

with DPPH and EPS.

108

Hydroxyl radical (OH) scavenging activity

109

Hydroxyl radical scavenging activity of EPS was evaluated as explained by Ye et al.

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(2012). The reaction mixture consists of 2.0 mL of 0.15 mM PBS (pH 7.4), 0.2 mL safranin T

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(0.52 mg/ mL), 1.0 mL of 6 mM EDTA-Fe(II), 0.8 mL of 6% (v/v) H2O2 and 1.0 mL EPS at

112

various concentrations. After incubation for 30 min at 40 °C, the absorbance (520 nm) was

113

evaluated against Ascorbic acid as a positive control. The scavenging activity % =

1 − 𝐴𝑆𝑎𝑚𝑝𝑙𝑒 − 𝐴𝐵𝑙𝑎𝑛𝑘 𝐴𝐶𝑜𝑛𝑡𝑟𝑜𝑙

× 100

114 115 116

6

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

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where, Asample - absorbance of the reagent mixture with EPS, Ablank - absorbance of

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the reagent mixture without EPS, and Acontrol - absorbance of the reagent mixture without EPS

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and H2O2.

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Fe-chelating activity

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Fe2+ chelating activity of EPS was investigated as described by Decker and Welch

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(1990) with slight modifications. Briefly, 10 µL of EPS at various concentrations was mixed

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with 0.5 mL FeCl2 (2 mM) and 1 mL ferrozine solution (5 mM) and kept for 20 minutes

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under dark conditions. The absorbance (562 nm) of the reactant solution was recorded along

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with distilled water as a control. Na2EDTA was used as a positive control. Fe2+Chelating activity % =

AControl − ATest × 100 AControl

126 127

where, Acontrol was the absorbance of the reagent without EPS, and Atest was the

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absorbance of the EPS.

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Statistical analysis

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All experiments were done in triplicates and the results were expressed as mean ± SD.

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Data processing was done using Microsoft office excel 2016 with Windows (2010) computer

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software.

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Results and Discussion

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Growth kinetics of L. lactis KC117496

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To optimize the growth condition of L. lactis KC117496, various trials was carried

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out. Fig.1 shows the growth pattern of L. lactis KC117496 at different shaking conditions

137

(100, 125 and 150 rpm). The optical density (O.D) values of 125 rpm (1.59) was higher

138

compared to 100 rpm (1.31) and 150 rpm (1.22), due to shear effect of bacterial cells in

139

shaker flask at 30 °C upto 48 h.

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

140

The exponential growth rate (0.082, 0.087, 0.076) and doubling time or generation

141

time (8.44, 7.87, 9.05 h) of L. lactis was observed at 100, 125 and 150 rpm, respectively. The

142

highest EPS production was recorded at 125 rpm (1.80 g/L) rather than 100 (0.740 g/L) and

143

150 rpm (0.340 g/L). In an earlier report, Leuconostoc mesenteroides NCIM 2947 isolated

144

from idli batter showed a growth rate of 0.2 and doubling time of 5 h (Subathra Devi et al.

145

2014).

146

Addition of sugars to increase the yield of EPS by L. lactis KC117496

147

To improve the EPS production, L. lactis KC117496 was grown in MRS medium

148

with 2% sucrose at 125 rpm and 30 °C. Addition of various disaccharides (sucrose, maltose

149

and lactose) at 2 % induced EPS production of L. lactis from 8 h onwards (Fig. 2). Highest

150

EPS production was observed with sucrose at 40 h of incubation. The yield of EPS is

151

correlated with specific gravity (S.G.) of the broth at each 8 h time interval. The S.G. and

152

yield of EPS at 40 h was improved more with sucrose (1.043; 1.94 g/L) compared to maltose

153

(1.036; 1.32 g/L) and lactose (1.028; 1.11 g/L).

154

Furthermore, supplementation of monosaccharides (glucose, fructose and galactose)

155

with 2 % sucrose was carried out for EPS production and S.G. The specific gravity (1.066)

156

and yield of EPS (4.55 g/L) with glucose addition was higher compared to fructose (1.053;

157

3.47 g/L) and galactose (1.060; 3.60 g/L). Under optimized conditions, the EPS production

158

was found to be increased compared to normal conditions (0.360 g/L) (Saravanan and Shetty

159

2016). Similarly, the yield of EPS from Weissella cibaria GA44 was improved through

160

media optimization up to 4.80 g/L (Adesulu-Dahunsi et al. 2018). Different sucrose

161

concentration (2, 5, 10, 15, 20, 25 and 30 %) was also tested for growth and EPS production.

162

The effect of increasing sucrose concentration was directly proportional to EPS production

163

whereas inversely proportional to the growth of the culture due to substrate inhibitory effect

164

(Supplement 1).

8

165 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

Nuclear magnetic resonance spectroscopy analysis

166

NMR is a spectroscopic technique to determine the structure of organic molecules.

167

The influence of structural character on chemical shifts and coupling constants is obtained

168

from correlation spectroscopy (COSY), hetero-nuclear single quantum coherence (HSQC)

169

and hetero-nuclear multiple bond correlation (HMBC) (Bubb 2003). Earlier studies on H1 and

170

13

171

Shetty 2016). Though heteronuclear, HSQC spectra (Fig. 3) showed the back bone as

172

(H1/C1) δ 5/98, (H2/C2) δ 3.6/72, (H3/C3) δ 3.7/73, (H4/C4) δ 3.5/70, (H5/C5) δ 3.9/71 and

173

(H6/C6) δ 4,3.75/66. These results correlate well with data published by Capek et al. (2011)

174

for Leuconostoc garlicum PR. Hetero-nuclear multiple bond correlation (HMBC) spectrum

175

displayed the cross signal between C1/H6 (98/3.8) and C6/H1 (66/5) signifying the existence

176

of α-1→6 linkage and cross signal between C1/H3 (98/3.7) and C3/H1 (73/5) indicating the

177

presence of α-1→6 linkage (Fig. 4). Similar, α-1→6 linkage was observed with galactan EPS

178

of Weissella confusa KR 780676 from an acidic fermented food (Kavitake et al. 2016).

179

MALDI-TOF-MS analysis

C NMR showed the EPS is a glucan comprises α-1→6 and α-1→3 linkages (Saravanan and

180

The glucan molecular weight was revealed through MALDI-TOF analysis (Fig. 5).

181

Depending upon the calibration curve of elution retention time of several standard

182

polysaccharides with glucose subunits, the molecular mass of glucan was estimated to be

183

~4.428 x 103 kDa. Similarly, Liu et al. (2017) also stated EPS from Lactobacillus plantarum

184

WLPL04 with a molecular weight of 6.61 × 104 Da.

185

DPPH radical scavenging activity

186

The DPPH free radical acts as an extensively proven tool for assessing the free radical

187

scavenging activities of antioxidants. DPPH free radical scavenging activity was resolved

188

through spectrophotometric analysis in which the antioxidants transfers an electron or a

189

hydrogen atom to DPPH, thereby neutralizing its free radical characteristic to form colour

190

solution (Liu et al. 2011). In this present work, the scavenging activity of glucan was 9

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

191

comparably in increasing drift with concentration (Fig. 6a). The scavenging activity at

192

various concentrations (100-500 µg/mL) of Vc (ascorbic acid) and glucan showed an

193

increasing trend of 2.8-75% and 6.8-74%, respectively. Previously, Li et al. (2014) and

194

Zhang et al. (2013) reported glucan EPS with high radical scavenging activity which was

195

41.92% and 52.23 %, respectively.

196

Hydroxyl radical (OH) scavenging activity

197

Hydroxyl radicals are extremely strong oxidants that easily react with bio-molecules

198

of living cells and creates destruction to the contiguous macromolecules in biological system

199

(Valko et al. 2016). The hydroxyl radical scavenging effect of both ascorbic acid and glucan

200

were increased with increasing concentrations from 100-500 µg/mL (Fig 6b). The hydroxyl

201

radical scavenging activity of Vc and glucan were 41.16-98.63% and 27.5-97.8%,

202

respectively. The scavenging activities of crude and purified fraction of EPS were 31.67%,

203

38.61, and 43.17% at 4.0 mg/mL (Liu et al. 2010).

204

Fe-chelating activity

205

The metal chelating activity plays a critical function as an antioxidant in the

206

biological system. The transition Fe2+ can influence lipid peroxidation through producing

207

hydroxyl radicals by fenton reaction and speed up lipid peroxidation through disintegrating

208

lipid hydroperoxides into alkoxyl and peroxyl radicals (Benedet and Shibamoto 2008). In this

209

study, Fe2+ chelating activity was observed from 100-500 µg/mL for both control (EDTA)

210

and glucan (Fig. 6c). Increase in chelating ability was increased with concentrations and

211

activity of glucan was less (5.8-72.5%) than control. Similar results were observed with crude

212

and two EPS fractions were 92.4%, 81.1% and 86.5% (Liu et al. 2011).

213

Conclusion

214

The L. lactis KC117496 produced glucan homopolysaccharide in MRS medium

215

supplemented with sucrose, having a molecular weight of ~4.428 x 103 kDa. Addition of

216

glucose along with sucrose at 2 % increased the yield up to 4.55 g/L. Structural investigation 10

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

217

of EPS through HMBC showed presence of α-1→6 and α-1→3 linked glucose subunits.

218

Antioxidant properties of glucan EPS exhibited an increasing trend at higher concentrations

219

for DPPH and Hydroxyl radical activity, while a lower metal chelating activity. These results

220

suggest that the glucan have potent antioxidant activities that could contribute as a natural

221

agent for possible application in functional foods and therapeutics.

222

Conflict of interest

223

The authors declare that they have no conflict of interest.

224

Acknowledgement

225

The authors are grateful to Pondicherry University for providing Sophisticated Analytical

226

Instrumental Facility.

227 228

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Figure captions Fig. 1. Growth curve of Leuconostoc lactis at different RPM (100,125,150).

Fig. 2. Optimization of different sugars to increase the yield of EPS from Leuconostoc lactis. EPS production (a) and the specific gravity (b) from different diasaccharides. Whereas, EPS production (c) and the specific gravity (d) using combination of sucrose and other monosaccharides. All the experiment was done in triplicate and results expressed as mean±SD.

Fig. 3. COSY spectrum indicating the cross signal between the protons and HSQC spectra showed the distribution of carbon and hydrogen in pyranose ring of monosaccharide units. Fig. 4. HMBC spectra revealed the α-1-6 linkage through the cross signal between H1/C6 and C1/H6. Fig. 5. MALDI-TOF-MS analysis of exopolysaccharide produced by Leuconostoc lactis.

Fig. 6. DPPH radical scavenging activity of EPS (A), Hydroxyl radical scavenging activity of EPS (B), Metal chelating activity of EPS (C). Vc- Ascorbic acid, C = Na2EDTA, EPSExopolysaccharide. Results are expressed as means ± standard deviations (n= 3).

15

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Fig. 1

16

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

Fig. 2

17

Fig. 3 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

18

Fig. 4 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

19

Intens. [a.u.]

Fig. 5

4428.6

600

500

4538.7

400

4583.4

300

200

3966.1

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

100

0

4000

6000

8000

10000

12000

14000 m /z

20

Fig. 6 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

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