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.
Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporation
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,
16
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
18
Indian traditional fermented foods are rich source of Lactic Acid Bacteria (LAB)
19
being widely consumed as a healthy diet with numerous benefits such as increased
20
digestibility, enhanced flavour, improved nutritional and pharmacological values. Idli is one
21
of the well-known, easily digestible, Indian traditional fermented food prepared from rice and
22
black gram dhal. Most of the LAB involved in fermentation of idli batter are capable of
23
producing exopolysaccharide (EPS) that are known to improve the texture and sensory
24
properties of idli (Prajapati, 2013; Patel et al. 2014; Saravanan et al. 2015). Furthermore, the
25
EPS has wide application as bio-flocculants, stabilizers, emulsifiers, bio-absorbents, drug
26
delivery agents and heavy metal removing agents (Liu et al. 2010). Dextran is a
27
homopolysaccharide which is produced by various LAB, especially Leuconostoc and
28
Streptococcus sp. in sucrose containing media (Kim et al. 2003). Glucan EPS produced by
29
Leuconostoc garlicum PR encompasses 95% α-1→6 glucopyranose linkage along with low
30
content of α-1→2, α-1→3 and α-1→4 linkages (Capek et al. 2011). Leuconostoc
31
pseudomesenteroides XG5 produced water soluble EPS composed of glucose subunits with
32
α-(1-6) linkage (Dextran) and the molecular mass was 2.6 × 103 kDa (Zhou et al. 2017).
33
Molecular weight of EPS produced by Leuconostoc citreum-BMS, L. mesenteroides-TMS,
34
Pediococcus pentosaceus-DPS and L. pseudomesenteroides-CM were around 106 Da (Abid et
35
al. 2017). Joshi and Koijam (2014) isolated L. lactis from ethnically fermented beverage
36
(Kyiad pyrsi) that yields EPS at a maximum of 0.340 g/L. Under normal conditions, EPS
37
produced from LAB ranged between 100-196 mg/L, while homopolysaccharides produced
38
are less than 1g/L. EPS produced by L. lactis and L. mesenteroides isolated from raw milk
39
ranges between 0.8–0.9 g/L (Van der Meulen et al. 2007). Under optimized conditions,
40
glucan production by L. dextranicum NRRL B-1146 reached a maximum of 1.063 g/L
41
(Majumder et al. 2009). EPS produced by Bacillus tequilensis FR9 showed potential strong
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
42
antioxidant effects through scavenging reactive oxygen species (ROS) (Rani et al. 2017). The
43
commercial glucan with different molecular weight (1, 4, and 14.7 × 104 Da) synthesized by
44
L. mesenteroides exhibited strong antioxidant, anticoagulant and immunomodulatory
45
activities (Soeiro et al. 2016). The in vitro antioxidant properties of the EPS showed
46
remarkable scavenging effects on superoxide anion radical and hydroxyl radical (Adesulu-
47
Dahunsi et al. 2018). Furthermore, EPS has been intensively employed as a food additive in
48
texture improvement which impart the development of innovative food products with
49
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
51
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
53
antioxidant property of α-D-glucan EPS has been studied to improve its production and
54
bioactive potential.
55
Materials and Methods
56
Microbial culture and chemicals
57
The EPS producing strain Leuconostoc lactis KC117496 used in this study was
58
isolated from naturally fermented idli batter. All reagents used were of analytical grade.
59
Growth condition of Leuconostoc lactis KC117496
60
For EPS production, 10 mL of L. lactis grown in MRS broth for 24 h (1x106 cfu/mL)
61
was used as an inoculum in 100 mL MRS broth supplemented with 2% sucrose. Flasks were
62
kept for incubation at 30 °C under different shaking conditions (100, 125 and 150 rpm) up to
63
48 h. At every 8 h intervals, optical density (O.D.) was assessed at 600 nm by using
64
spectrophotometer (UV-1800, Shimadzu, North America) and growth curves were plotted.
65
Extraction and purification of EPS
66
At the time of harvesting, 48 h grown culture broth was subjected to 100 °C for 10
67
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
68
temperature, the biomass was removed by centrifugation at 4100 xg for 20 min. The resulting
69
supernatant was treated with 2 % Trichloroacetic acid to remove the proteinaceous materials
70
by centrifugation. Then EPS was precipitated using thrice the volume of ice cold ethanol and
71
left overnight. The precipitated EPS was separated via centrifugation (19200 xg, 15 min) and
72
dissolved in Milli Q water. For further purification, EPS was encased in a dialysis bag (12-14
73
kDa) and dialysed until 48 h at 4 °C in Milli Q water (Saravanan and Shetty 2016). Dialyzed
74
EPS was subjected to lyophilization. Quantification of EPS was done using phenol sulphuric
75
acid method (Dubois et al. 1956).
76
Optimization kinetics of L. lactis KC117496 to increase EPS production
77
The batch fermentation was tested initially for EPS production using different
78
disaccharides such as sucrose, lactose and maltose as sole carbon source (2 % w/v).
79
Subsequently, the disaccharide that yields the highest EPS was further combined with
80
monosaccharide (2 % w/v) such as glucose, mannose and galactose after 8 h of incubation.
81
Freshly prepared 10 % inoculum (24 h) of L. lactis was added into each flask. EPS
82
production was observed at every 8 h interval up to 48 h under shaking conditions.
83
Nuclear magnetic resonance spectroscopy analysis
84
In structural analysis, 1H and
13
C NMR spectrum was measured using the partially
85
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
90
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
92
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
97
laser repetition rate 2000 Hz.
98
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
102
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
104
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.
110
(2012). The reaction mixture consists of 2.0 mL of 0.15 mM PBS (pH 7.4), 0.2 mL safranin T
111
(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
117
where, Asample - absorbance of the reagent mixture with EPS, Ablank - absorbance of
118
the reagent mixture without EPS, and Acontrol - absorbance of the reagent mixture without EPS
119
and H2O2.
120
Fe-chelating activity
121
Fe2+ chelating activity of EPS was investigated as described by Decker and Welch
122
(1990) with slight modifications. Briefly, 10 µL of EPS at various concentrations was mixed
123
with 0.5 mL FeCl2 (2 mM) and 1 mL ferrozine solution (5 mM) and kept for 20 minutes
124
under dark conditions. The absorbance (562 nm) of the reactant solution was recorded along
125
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
128
absorbance of the EPS.
129
Statistical analysis
130
All experiments were done in triplicates and the results were expressed as mean ± SD.
131
Data processing was done using Microsoft office excel 2016 with Windows (2010) computer
132
software.
133
Results and Discussion
134
Growth kinetics of L. lactis KC117496
135
To optimize the growth condition of L. lactis KC117496, various trials was carried
136
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.
7
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
References
229 230
Abid Y, Casillo A, Gharsallah H, Joulak I, Lanzetta R, Corsaro MM, Azabou S (2017)
231
Production and structural characterization of exopolysaccharides from newly isolated
232
probiotic lactic acid bacteria. Int J Biol Macromol 108: 719-728.
233
Adesulu-Dahunsi AT, Sanni AI, Jeyaram K (2018) Production, characterization and In vitro
234
antioxidant activities of exopolysaccharide from Weissella cibaria GA44. LWT - Food
235
Sci Technol 87: 432–442.
236 237 238 239
Benedet JA, Shibamoto T (2008) Role of transition metals, Fe(II), Cr(II), Pb(II), and Cd(II) in lipid peroxidation. Food Chem 107: 165-168. Bubb WA (2003) NMR spectroscopy in the study of carbohydrates: Characterizing the structural complexity. Concepts Magn Reson part A Bridg Educ Res 19: 1-19.
11
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
240
Capek P, Hlavoňová E, Matulová M, Mislovicová D, Růžička J, Koutný M, Keprdová L
241
(2011) Isolation and characterization of an extracellular glucan produced by
242
Leuconostoc garlicum PR. Carbohydr Polym 83: 88-93.
243
De Vuyst L, De Vin F, Vaningelgem F, Degeest B (2001) Recent developments in the
244
biosynthesis and applications of heteropolysaccharides from lactic acid bacteria. Int
245
Dairy J 11: 687-707.
246 247 248 249
Decker EA, Welch B (1990) Role of Ferritin as a Lipid Oxidation Catalyst in Muscle Food. J Agric Food Chem 38: 674-677. Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith F (1956) Colorimetric Method for Determination of Sugars and Related Substances. Anal Chem 28: 350-356.
250
Ismail B, Nampoothiri KM (2010) Production, purification and structural characterization of
251
an exopolysaccharide produced by a probiotic Lactobacillus plantarum MTCC 9510.
252
Arch Microbio 192: 1049-1057.
253
Joshi SR, Koijam K (2014) Exopolysaccharide production by a lactic acid bacteria,
254
Leuconostoc lactis isolated from ethnically fermented beverage. Natl Acad Sci Lett 37:
255
59-64.
256
Kavitake D Devi PB, Singh SP, Shetty PH (2016) Characterization of a novel galactan
257
produced by Weissella confusa KR780676 from an acidic fermented food. Int J Biol
258
Macromol 86: 681-689.
259
Kim D, Robyt JF, Lee SY, Lee JH, Kim YM (2003) Dextran molecular size and degree of
260
branching as a function of sucrose concentration, pH, and temperature of reaction of
261
Leuconostoc mesenteroides B-512FMCM dextransucrase. Carbohydr Res 338: 1183-
262
1189.
12
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
263
Li W, Ji J, Chen X, Jiang M, Rui X, Dong M (2014) Structural elucidation and antioxidant
264
activities of exopolysaccharides from Lactobacillus helveticus MB2-1. Carbohydr
265
Polym 102: 351-359.
266 267
Liu C, Lin Q, Gao Y, Ye L, Xing Y, Xi T (2007) Characterization and antitumor activity of a polysaccharide from Strongylocentrotus nudus eggs. Carbohydr Polym 67: 313-318.
268
Liu C, Lu J, Lu L, Liu Y, Wang F, Xiao M (2010) Isolation, structural characterization and
269
immunological activity of an exopolysaccharide produced by Bacillus licheniformis 8-
270
37-0-1. Bioresour Technol 101: 5528-5533.
271
Liu CF, Tseng KC, Chiang SS, Lee BH, Hsu WH, Pan TM (2011) Immunomodulatory and
272
antioxidant potential of Lactobacillus exopolysaccharides. J Sci Food Agric 9: 2284-
273
2291.
274
Liu Z, Zhang Z, Qiu L, Zhang F, Xu X, Wei H, Tao X (2017) Characterization and
275
bioactivities of the exopolysaccharide from a probiotic strain of Lactobacillus
276
plantarum WLPL04. J Dairy Sci 100: 6895-6905.
277
Majumder A, Singh A, Goyal A (2009) Application of response surface methodology for
278
glucan production from Leuconostoc dextranicum and its structural characterization.
279
Carbohydr Polym 75: 150-156.
280
Patel A, Prajapati JB, Holst O, Ljungh A (2014) Determining probiotic potential of
281
exopolysaccharide producing lactic acid bacteria isolated from vegetables and
282
traditional Indian fermented food products. Food Biosci 5: 27-33.
283 284
Prajapat AP (2013) Food and Health Applications of Exopolysaccharides produced by Lactic acid Bacteria. Adv Dairy Res 1-8.
285
Rani RP, Anandharaj M, Sabhapathy P, Ravindran AD (2017) Physiochemical and biological
286
characterization of novel exopolysaccharide produced by Bacillus tequilensis FR9
287
isolated from chicken. Int J Biol Macromol 96: 1-10.
13
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
288
Saravanan C, Gopu V, Shetty PKH (2015) Diversity and functional characterization of
289
microflora isolated from traditional fermented food idli. J Food Sci Technol 52: 7425-
290
7432.
291
Saravanan C, Shetty PKH (2016) Isolation and characterization of exopolysaccharide from
292
Leuconostoc lactis KC117496 isolated from idli batter. Int J Biol Macromol 90: 100-
293
106.
294
Soeiro VC, Melo KRT, Alves MGCF, Medeiros MJC, Grilo MLPM, Almeida-Lima J, …
295
Rocha HAO (2016) Dextran: Influence of molecular weight in antioxidant properties
296
and immunomodulatory potential. Int J Mol Sci 17: 1340.
297 298
Subathra Devi C, Reddy S, Mohanasrinivasan V (2014) Fermentative production of dextran using Leuconostoc spp. isolated from fermented food products. Front Biol 9: 244-253.
299
Valko M, Jomova K, Rhodes CJ, Kuča K, Musílek K (2016) Redox-and non-redox-metal-
300
induced formation of free radicals and their role in human disease. Arch Toxicol 90: 1-
301
37.
302
Van der Meulen R, Grosu-Tudor S, Mozzi F, Vaningelgem F, Zamfir M, de Valdez GF, De
303
Vuyst L (2007) Screening of lactic acid bacteria isolates from dairy and cereal
304
products for exopolysaccharide production and genes involved. Int J Food Microbiol
305
118: 250-258.
306
Yang B, Wang J, Zhao M, Liu Y, Wang W, Jiang Y (2006) Identification of polysaccharides
307
from pericarp tissues of litchi (Litchi chinensis Sonn.) fruit in relation to their
308
antioxidant activities. Carbohydr Res 341: 634-638.
309
Ye S, Liu F, Wang J, Wang H, Zhang M (2012) Antioxidant activities of an
310
exopolysaccharide isolated and purified from marine Pseudomonas PF-6. Carbohydr
311
Polym 87: 764-770.
14
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
312
Zhang M, Tang X, Wang F, Zhang Q, Zhang Z (2013) Characterization of Lycium barbarum
313
polysaccharide and its effect on human hepatoma cells. Int J Biol Macromol, 61: 270-
314
275.
315
Zhou Q, Feng F, Yang Y, Zhao F, Du R, Zhou Z, Han Y (2017) Characterization of a dextran
316
produced by Leuconostoc pseudomesenteroides XG5 from homemade wine. Int J Biol
317
https://doi.org/10.1016/j.ijbiomac.2017.10.098.
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
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. 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
21
Supplementary Material
Click here to access/download
Supplementary Material Supplement 1.docx
Copyright Transfer Statement
