Subscriber access provided by University of Newcastle, Australia
Article
Structural Characterization and in vitro Fermentation of #-mannooligosaccharides Produced from Locust bean gum by GH-26 Endo-#-1,4-mannanase (ManB-1601) Praveen Kumar Srivastava, Deepesh Panwar, Harish Prashanth KV, and Mukesh Kapoor J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b00123 • Publication Date (Web): 22 Feb 2017 Downloaded from http://pubs.acs.org on February 24, 2017
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 36
Journal of Agricultural and Food Chemistry
1
Structural Characterization and in vitro Fermentation of β-mannooligosaccharides
2
Produced from Locust bean gum by GH-26 Endo-β-1,4-mannanase (ManB-1601)
3
Praveen Kumar Srivastava†, Deepesh Panwar†,§, K. V. Harish Prashanth‡,§ and Mukesh
4
Kapoor†,§,*
5
†
Department of Protein Chemistry and Technology; ‡Department of Biochemistry; CSIR-Central
6
Food Technological Research Institute, Mysuru-570 020, India; §Academy of Scientific and
7
Innovative Research (AcSIR), CSIR-CFTRI Campus, Mysuru, India
8
* Tel: +91-821-2515331, E-mail:
[email protected] 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
1 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 2 of 36
24
ABSTRACT
25
Size exclusion chromatography of β-mannooligosaccharides (β-MOS) mixture, obtained from
26
ManB-1601 hydrolysis of locust bean gum, resulted in separation of oligosaccharides with varied
27
degree of polymerization (DP 2, 3 and 5). The oligosaccharides were structurally [ESI-MS,
28
FTIR, XRD, TGA and NMR (1H and 13C)] and functionally (in vitro fermentation) characterized.
29
DP2 oligosaccharide was composed of two species (A) mannopyranose β-1,4 mannopyranose
30
and (B) α-1,6-galactosyl-mannopyranose, while DP3 oligosaccharide showed presence of only
31
one species i.e. α-D-galactosyl-β-D-mannobiose. ManB-1601 was capable of cleaving near the
32
branch points in the substrate resulting in oligosaccharides with galactose at the terminal position
33
apart from attacking unsubstituted β-1,4 glycosidic linkages. DP2 and DP3 improved the growth
34
of three out of seven species of Lactobacillus while, DP5 resulted in poor growth of all
35
Lactobacillus spp. under in vitro conditions. DP2, DP3 and DP5 were found to inhibit the growth
36
of E. coli, L. monocytogenes and S. typhi.
37
Keywords: GH-26 endo-β-1,4-mannanase, Lactobacillus spp., β-mannooligosaccharides,
38
Structural characterization, in vitro fermentation
39 40
41
42
43
44 45
2 ACS Paragon Plus Environment
Page 3 of 36
Journal of Agricultural and Food Chemistry
46
INTRODUCTION
47
Currently, several researchers and leading food companies are developing functional food
48
preparations
49
xylooligosaccharides, pecticoligosaccharides, isomaltooligosaccharides, galactooligosaccharides,
50
and fructooligosaccharides)/probiotics due the realization of the intricate relationship between
51
food ingredients, beneficial gut microflora and health.1-8
containing
prebiotics
(arabinooligosaccharides, arabinoxylooligosaccharides,
52
Mannooligosaccharides (MOS), including α-MOS and β-MOS, are a relatively new class
53
of oligosaccharides, that have gained significant interest as a prebiotic.9,10 β-MOS are not acted
54
upon by gastric or pancreatic enzymes and are used by a selected group of beneficial gut
55
microflora for inducing several physiological changes.11 β-MOS can be produced by hydrolytic
56
cleavage of mannans by endo-β-1,4-mannanase (EC 3.2.1.78) and show variability with respect
57
to their degree of polymerization and presence/position of substituents.12-14 Recently, a number
58
of reports have cited the prebiotic potential of crude β-MOS from guar gum, locust bean gum,
59
konjac mannan and copra meal using endo-β-1,4-mannanase.10, 15-19 However, detailed studies
60
encompassing purification, structural characterization and in vitro fermentation behaviour of β-
61
MOS on probiotic populations need to be carried out in order to provide further information.
62
ManB-1601 (UniProtKB-A0A0A1E3J1) obtained from Bacillus sp. CFR1601 is a GH26,
63
thermostable, endo-β-1,4-mannanase having (β/α)8-barrel spatial arrangement.20-21 In the present
64
study, attempts were made to understand the molecular properties [degree of polymerization,
65
composition of monomer units, chemical structure and thermal properties] of β-MOS derived
66
from locust bean gum after ManB-1601 hydrolysis; to analyze the cleavage pattern of ManB-
67
1601 on locust bean gum and to evaluate the propensity of β-MOS towards fermentation by
68
Lactobacillus spp. and food borne pathogens.
3 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 4 of 36
69
MATERIALS AND METHODS
70
Microbial culture and chemicals
71
Locust bean gum, volatile free acid standard mix, fructo-oligosaccharides (FOS) [composed of
72
glucose-(fructose)n with β−2→1 linkage between the fructose monomer units, fructose chain
73
length: 2 to 60, average DP>10] and 3,5-dinitrosalicylic acid were purchased from Sigma-
74
Aldrich (St. Louis, MO). Biogel-P2 was purchased from Bio-Rad Laboratories (Hercules, CA).
75
Commercial mannobiose having degree of polymerization of two (CDP2) was purchased from
76
Megazyme, Ireland. Microbial culture media and other ingredients were procured from Himedia
77
(Bengaluru, India). Lactobacillus cultures were obtained from National Culture Collection of
78
Industrial Microorganisms (NCIM) at CSIR-National Chemical Laboratory (NCL), Pune (India).
79
Cultures of food borne pathogens (Escherichia coli ATCC 11775, Salmonella typhi ATCC
80
25241 and Listeria monocytogenes ATCC 13932) were obtained from Microbiology and
81
Fermentation Technology Department at CSIR-CFTRI, Mysuru. All other reagents used were of
82
the highest purity available commercially.
83
Production of β-MOS from locust bean gum using ManB-1601
84
ManB-1601 was produced and purified as per the protocol described earlier.20,21 For the
85
preparation of β-MOS, 1% (w/v) locust bean gum in 50 mM sodium phosphate buffer (pH 7, 80
86
mL) was incubated with purified ManB-1601 (100 U/mL) under shaking conditions (200 rpm) at
87
50 oC for 270 min.18 The hydrolysate obtained was kept in boiling water bath for 5 min to
88
inactivate the enzyme. Enzyme resistant and insoluble fractions were removed by centrifugation
89
at 3622 x g for 10 min at room temperature. Thereafter, three volumes of ethanol were added to
90
the supernatant for precipitating high-molecular-mass fractions and centrifuged at 3622 x g for
4 ACS Paragon Plus Environment
Page 5 of 36
Journal of Agricultural and Food Chemistry
91
20 min. The supernatant containing β-MOS was flash evaporated and stored at -20 oC until
92
further use.18
93
Purification of β-MOS
94
β-MOS mixture obtained after locust bean gum hydrolysis by ManB-1601 was fractionated on
95
Biogel P-2 (bed volume, 100 mL) previously equilibrated with Milli-Q water at room
96
temperature in accordance to our previous report.18 Elution was done using Milli-Q water at a
97
flow rate of 10 mL/h.
98
Characterization of purified β-MOS
99
Electron spray ionization- mass spectrometry (ESI-MS)
100
The molecular mass of purified β-MOS fractions was determined by ESI-MS under the
101
following operational conditions: capillary voltage 3.5 kV, core voltage 100 V, source
102
temperature 120 oC, desolvation temperature 300 oC, gas (nitrogen) 500 L/h and core gas (argon)
103
50 L/h.
104
Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD)
105
IR spectra of β-MOS were determined using an IFS 25 FTIR spectrophotometer (Bruker,
106
Ettlingen, Germany). β-MOS were ground and mixed with spectroscopic grade potassium
107
bromide powder (KBr) and pressed into a pellet. The spectra of the samples were recorded in the
108
range of 700-1500 cm-1. The XRD patterns of β-MOS were obtained using a SmartLab 3 kW X-
109
ray diffractometer (Rigaku, Tokyo, Japan) with Cu Kα source at 2θ angle probed 5-75o at 0.02
110
/min scanning speed.
111
Thermogravimetric and differential thermogravimetric analysis
112
Thermogravimetric (TGA) and differential thermal (DTA) analysis of β-MOS were carried out
113
under continuous N2 flow on a STA 2500 Regulus differential scanning calorimeter (Netzsch
5 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 6 of 36
114
Instruments, Burlington, MA). Samples were weighed (~2-5 mg) and heated from 30-700 oC at a
115
heating rate of 10 oC/min in aluminium pans. The empty pan was used as reference.
116
1
H and 13C NMR spectroscopy
117
The NMR spectroscopic studies were carried out on an Avance AQS 500MHz
118
spectrophotometer (Bruker, Reinstetten, Germany) NMR spectrometer. 5 mg of sample was
119
dissolved in 600µl D2O and spectra were collected. 1H spectrum was collected at 500 MHz with
120
spectral width kept at 10330 Hz. The signal for water occurs at 4.8 ppm frequency in 1H NMR
121
spectra on the D2O solvent used. The water suppression pulse program zgpr was employed to
122
collect the 1H spectrum with 16 scans. The
123
10K scans.
124
In vitro fermentation of β-MOS by lactobacilli and food borne pathogens
125
Seed inoculum
126
Seed inocula of Lactobacillus sp. [L. plantarum (NCIM 2372), L. fermentum (NCIM 2165), L.
127
casei var. rhamnosus (NCIM 2125), L. brevis (NCIM 2090), L. acidophilus (NCIM 2285), L.
128
casei (NCIM 2126) and L. helveticus (NCIM 2126)] and food borne pathogens [(E. coli (ATCC
129
11775), S. typhi (ATCC 25241) and L. monocytogenes (ATCC 13932)] were prepared by
130
cultivation in MRS (de Man, Rogosa and Sharpe) medium under static conditions and tryptic soy
131
broth (TSB) under shaking (200 rpm) conditions, respectively for 12h at 37 oC.
132
Batch fermentation
133
Batch fermentation studies for Lactobacillus spp. and food borne pathogens were carried out in
134
MRS and TSB media (devoid of glucose, 1 mL), respectively. The media were supplemented
135
separately with either filter sterilized (0.22µ) β-MOS having degree of polymerization (DP2,
136
DP3, DP5 and CDP2), FOS (positive control-1) or glucose (positive control-2) at a final
13
C NMR spectra were collected at 125 MHz with
6 ACS Paragon Plus Environment
Page 7 of 36
Journal of Agricultural and Food Chemistry
137
concentration of 0.1% (w/v). Thereafter, MRS media was inoculated with 10 µl (A600nm~0.2) of
138
inoculum obtained from respective Lactobacillus spp. and incubated under static conditions (37
139
o
140
respective food borne pathogens and incubated under shaking conditions (37 oC, 24h, 200 rpm).
141
The fermented media was centrifuged (3500 rpm, 5 min, RT) and cells were resuspended in
142
sterile phosphate buffer saline. Growth characteristics of the cultures were measured by
143
determining the log colony forming units (CFU)/mL, absorbance (A600nm) and pH.
144
SCFA analysis
145
The cell free supernatants of Lactobacillus spp. were analysed for short chain fatty acid (SCFA)
146
analysis. 100 mg of sodium chloride was added to each culture supernatant (450 µl) and the
147
mixture was acidified by the addition of sulphuric acid (125 µl, 9 M). The acidified mixture was
148
extracted by an equal volume of diethyl ether. 1 µl of sample (top layer) was subjected to gas
149
chromatography on a Clarus 580 instrument (Perkin Elmer, Waltham, MA). The column used
150
was a 30 m x 0.32 mm i.d., film thickness 0.25 µm, Elite-WAX cross bond- PEG (Perkin Elmer,
151
Waltham, MA). Column conditions were 60 oC (2 min hold), 2 oC/min to 135 oC (0.5 min hold).
152
Injector and detector were kept at 220 and 230 oC, respectively. Volatile free acid standard mix
153
was used as the standard (Sigma-Aldrich, St. Louis, MO).
154
Analytical procedures
155
ManB-1601 activity was assayed by determining the release of reducing sugar equivalents from
156
locust bean gum (0.5% w/v) in 50 mM sodium phosphate buffer (pH 7) at 55 oC using the
157
dinitrosalicylic acid method.22 One unit of ManB-1601 activity was defined as the quantity of
158
enzyme required for liberation of 1.0 µmol mannose/minute under the standard assay conditions.
C, 48h), while TSB media was inoculated with 10 µl (A600nm~0.2) of inoculum obtained from
7 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 8 of 36
159
Total carbohydrate content of β-MOS was determined as glucose equivalents by the phenol-
160
sulfuric acid method with slight modifications.23
161
Statistical analysis
162
Measurements pertaining to in vitro fermentation experiments (A600nm and pH) were carried out
163
in duplicates and presented as arithmetic mean. Calculation for arithmetic mean was done using
164
using Sigma plot 10.0. Analysis of variance (ANOVA) of data was performed with Origin Pro 7
165
(Origin Lab Corporation, Northampton, MA) statistical software using Tuckey’s test at 0.01
166
level.
167
RESULTS AND DISCUSSION
168
During the last two decades, non-digestible prebiotic oligosaccharides have garnered increased
169
attention from both industry and academia due to their positive impact on the gut microbiome.4 It
170
has been shown conclusively that DP and molecular structure of these prebiotic oligosaccharides
171
play a vital role in their utilization by probiotic bacteria.24 Therefore, the overall aim of the
172
present study was to establish the structure (glycosidic linkages, oligosaccharide composition
173
and DP)-function (in vitro fermentability) relationship of β-MOS with probiotic Lactobacillus
174
spp. and/or food borne pathogens.
175
Production, purification and characterization of β-MOS
176
Hydrolysis of locust bean gum by ManB-1601 for 270 min resulted in generation of β-MOS with
177
an overall yield of 30% (w/v). Separation of crude β-MOS mixture by Biogel-P2
178
chromatography resulted in five well separated peaks which were designated as P1, P2, P3, P4,
179
and P5 (Figure 1A). Peak P1-P5 were identified as cationised molecules [M+Na]+ by ESI-mass
180
spectroscopy (Figure 1B). The P1 and P2 peaks corresponded to a pentasaccharide (DP5) [P1-
181
599, derived from cross cleavage of branched pentasaccharide; 25 P2- 851, [{(180x 5) - (4x18)} +
8 ACS Paragon Plus Environment
Page 9 of 36
Journal of Agricultural and Food Chemistry
182
23]. Peaks P3 (DP3), P4 (DP2) and P5 (monomer) corresponded to trisaccharide [527, {(180x 3)
183
- (2x18)} + 23], disaccharide [365, {(180x 2) - (1x18)} + 23]; 707, two disaccharide residues
184
with one sodium (342 + 342+23)] and mannose [203, (180 + 23)], respectively. The extent of
185
hydrolysis of galactomannans by endo-β-1,4-mannanase derived either from legume seeds or
186
microorganisms like fungi and bacteria and subsequent generation of oligosaccharides has been
187
shown to be dependent noticeably on
188
galactomannan and endo-β-1,4-mannanase family [as endo-β-1,4-mannanase from different
189
families display different catalytic properties towards substituted mannans as seen in case of
190
xylanases.26-29 Cerqueira et al.30 reported presence of oligosaccharides of DP ranging from 2 to 7
191
after endo-β-1,4-mannanase hydrolysis of galactomannan obtained from Gleditsiatria canthos.
192
Ghosh et al.10 showed production of mannose, mannobiose and mannotriose after endo-β-1,4-
193
mannanase hydrolysis of copra meal. Similarly, endo-β-1,4-mannanase from Penicillium
194
occitanis was able to produce mannose, mannobiose, mannotriose and mannotetrose from locust
195
bean gum.31
196
FTIR spectroscopy and XRD
197
FTIR spectra of purified β-MOS were recorded in the carbohydrate region (700-1500 cm-1)
198
(Figure 2). Peaks at 816, 811 and 817 cm-1 found in disaccharide (DP2), trisaccharide (DP3) and
199
pentasaccharide (DP5) revealed the presence of anomeric configurations (α and β anomers). The
200
glycosidic linkage of α-D-galactopyranose and β- D-mannopyranose units in DP2, DP3 and DP5
201
β-MOS was observed at 875, 867 and 873 cm-1.32, 33 The peaks between 1037 to 1155 cm-1 in
202
DP2, DP3 and DP5 β-MOS signified the stretching vibration of C-O and C-O-H bonds present in
203
the glycosidic linkage of galactomannans.33, 34
frequency and pattern of galactose substitution in
9 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 10 of 36
204
XRD studies were carried out only for DP2 and DP3 β-MOS, as the hygroscopic nature
205
of DP5 β-MOS prevented the removal of bound water by lyophilisation and subsequent XRD
206
studies. The broad characteristic peak of less intensity at 20o in DP2 (Figure 3A) and DP3
207
(Figure 3B) β-MOS resulted due to more rotational freedom in the molecule, indicated their
208
typical non-crystalline structure and amorphous nature. Similar results were found in case of
209
chitosan oligosaccharides, konjac glucomannan-based films and guar gum.35-37
210
Thermogravimetric (TGA) and differential thermal (DTA) analysis
211
It is quintessential to understand the thermal behaviour of prebiotic molecules like β-
212
mannooligosaccharides as many times they are added in food products which undergo thermal
213
processing like roasting, frying and baking. In case, the prebiotic oligosaccharide is not able to
214
withstand high temperatures encountered during thermal processing it is highly unlikely that it
215
can deliver desirable attributes in the functional food. TGA and DTA is known to provide
216
important information like determining the contents of oligosaccharides from the residual mass
217
and also prediction for the ratio of bound water by applying mass loss calculation during first
218
decomposition.38 The TGA (Figure 3C) and DTA (Figure 3D) graphs revealed three stages of
219
mass loss for DP2 and DP3 β-MOS. TGA and DTA of DP5 were not carried out due to reasons
220
stated above. The first mass loss (up to 12%) for DP2 and DP3 β-MOS occurred at 150 and 171
221
o
222
loss (up to 52%) occurred at the main chain fracture temperatures i.e. 314 and 318 oC for DP2
223
and DP3 β-MOS, respectively.40 The final mass loss (up to 81%) of DP2 and DP3 β-MOS
224
occurred at 510 and 516 oC, respectively which represented the decomposition temperatures and
225
afterwards little residual mass of oligosaccharides was left.
C, respectively which might be due to loss of adsorbed or structural water.39 The second mass
226
10 ACS Paragon Plus Environment
Page 11 of 36
Journal of Agricultural and Food Chemistry
227
Nuclear Magnetic Resonance (NMR) analysis of DP2 and DP3
228
P4 (DP2 oligosaccharide) (Figure 4A and B) and P3 (DP3 oligosaccharide) (Figure 5A and B)
229
peaks were further analysed using NMR (500 MHz) for determination of their respective
230
structures. P1 and P2 peaks were not structurally characterized due to their poor yield. Empirical
231
rules for chemical shifts and comparative analysis of relative intensities of resonances were
232
applied for making assignments and the results obtained were in agreement with earlier
233
studies.12,13 1H and
234
species of oligosaccharides. A) Mannopyranose β-1,4-mannopyranose (structure 4, figure 6):
235
From 1H NMR spectra the disaccharide showed resonances at 5.2 ppm (MRα) and 4.7 ppm
236
(MNRβ) which can be assigned to the anomeric protons of the reducing and non-reducing terminal
237
mannose residues of mannopyranose β-1,4- mannopyranose, respectively.
13
C NMR spectra of DP2 oligosaccharides revealed the presence of two
238
From 13C NMR, presence of glycosidic bond (β-1,4) was shown by the resonance of C4
239
in reducing D-mannose at 81.1 ppm. However, in case of non-reducing D-mannose residue, C4
240
resonance appeared at 76.3 thereby confirming the presence of mannopyranose β-1,4-
241
mannopyranose. The spectrum of the disaccharide showed resonances at 91.9 ppm (MRα) and
242
103.4 ppm (MNRβ) which can be assigned to the anomeric carbons of the reducing and non-
243
reducing terminal mannose residues of mannopyranose β-1,4- mannopyranose, respectively. B)
244
α-1,6-Galactosyl-mannopyranose (structure 3, figure 6): From 1H NMR spectra, the chemical
245
shifts at 5.1 ppm and 4.6 ppm could be assigned to protons of galactose (H1) and mannose (H6)
246
at the branch point of α-1,6-galactosyl-mannopyranose (Table 1). From
247
showed two intense peaks around 93.6 and 99.9 ppm corresponding to C1 of D-mannose and D-
248
galactose residues, respectively (Table 2). The resonances corresponding to ring carbons C2, C3,
249
C4 and C5 are shown in Table 2. Interestingly, C6 δ value appeared around 66.4 ppm for D-
13
C NMR, this species
11 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 12 of 36
250
mannose and 60.7 ppm (down field shift) for D-galactose, respectively. In the latter case, solitary
251
C6 of D-galactose showed two resonances (α and β) (Table 2). Therefore, this disaccharide
252
product of ManB-1601 depolymerized locust bean gum (structure 1, figure 6) could be
253
concluded as α-1,6-galactosyl-mannopyranose. Considerable data was also obtained by spin-
254
echo Fourier Transform (SEFT) analysis, 2D NMR, HSQC and HMBC with respect to DP2
255
oligosaccharide so as to confirm the presence of two species of disaccharide (data not shown).
256
The ratio of the integrals for the anomeric protons (1H NMR) assigned to the non-reducing
257
residue to those assigned to the reducing residue was about ~1, as would be expected for a
258
disaccharide. The generation of appreciable quantities of α-1,6-galactosyl-mannopyranose
259
residues by ManB-1601, to the best of our knowledge, is the shortest substituted oligosaccharide
260
reported to date produced from a bacterial endo-β-1,4-mannanase. 1
261
H and 13C NMR spectrum of trisaccharide explicitly showed presence of α-D-galactosyl-
262
β-D-mannobiose (structure 2, figure 6) devoid of any other trisaccharide (Figure 5). The
263
assignments made for
264
Table 2. A careful analysis of Biogel P2 profile (Peak 5) and disaccharide NMR data indicate
265
that ManB-1601 might have the hydrolyzing capability of cleaving α-D-galactosyl-β-D-
266
mannobiose to D-mannose (structure 5, figure 6) and α-1,6-galactosyl-mannopyranose.
13
C NMR spectrum of α-D-galactosyl-β-D-mannobiose are indicated in
267
On the basis of the cleavage pattern of galactomannans or galactoglucomannan by endo-
268
β-1,4-mannanase reported to date, we suggest the following three broad groups of endo-β-1,4-
269
mannanase with respect to the type of oligosaccharides generated.
270
First group: Endo-β-1,4-mannanase which give rise to substituted oligosaccharides with
271
galactose at terminal position: can be formed with endo-β-1,4-mannanase from Aspergillus niger
272
28
and Penicillium purpurogenum 29 wherein the shortest substituted oligosaccharide obtained in
12 ACS Paragon Plus Environment
Page 13 of 36
Journal of Agricultural and Food Chemistry
273
hydrolysis of galactomannan was a trisaccharide [α-D-galactosyl-β-D-mannobiose (GallMan2),
274
endo-β-D-mannanase from Trichoderma reesei which produced GallMan2 (as the shortest
275
substituted oligosaccharide i.e. trisaccharide) from galactoglucomannan of pine kraft pulp 41 and
276
endo-β-1,4-mannanase from Bacillus subtilis, which had a more limited ability to hydrolyse
277
galactomannans and the shortest substituted product was α-D-galactosyl-β-D-mannotetrose (Gal1-
278
Man4) (mannopentose).27
279
Second group: Endo-β-1,4-mannanase which give rise to substituted oligosaccharides with
280
galactose at non-terminal position: can be formed of endo-β-1,4-mannanase from Aspergillus
281
niger which hydrolysed carob galactomannan to a series of D-galactose-containing β-D-
282
mannosaccharides with galactose substitution at non-terminal positions.28
283
Third group: Endo-β-1,4-mannanase which give rise to unsubstituted oligosaccharides: can be
284
formed of β-mannanase from Trichoderma reesei which showed release of un-substituted
285
mannose, mannotriose and mannopentose from locust bean gum,12 and β-D-mannanases from
286
lucerne seed which generated unsubstituted β-D-manno-biose, -triose, and -tetrose after the
287
hydrolysis of carob galactomannan.28
288
Most of the endo-β-1,4-mannanase reported to date might come under either of the above
289
second or third group as steric hindrance by α-1,6 linked galactose in mannans do not allow
290
endo-β-1,4-mannanase to cleave near branch points and consequently most of the generated
291
products are linear β-1,4 linked mannopyranose residues or β-1,4 linked mannopyranose residues
292
substituted with galactose at the non-terminal position(s).
293
A schematic representation of the proposed depolymerization pattern of ManB-1601 on
294
locust bean gum is shown in Figure 6 A and B on the basis of size-exclusion chromatography,
295
ESI-MS, and NMR spectroscopy of locust bean gum derived β-MOS. Accordingly, ManB-1601
13 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 14 of 36
296
might fall in the first group of classification mentioned above as it generates α-1,6-galactosyl-
297
mannopyranose residues by cleaving near the branch points in galactomannans (cleavage site
298
shown by hollow arrow), apart from attacking internal β-1,4 glycosidic linkages (cleavage site
299
shown by solid arrow) (Figure 6A).
300
In vitro fermentation of β-MOS by lactobacilli and food borne pathogens
301
The present study is the first report on prebiotic contributions of endo-β-1,4-mannanase derived β-
302
MOS from locust bean gum with defined DP and chemical structure. Purified β-MOS (DP 2, 3 and
303
5) produced from the hydrolysis of locust bean gum by ManB-1601 were utilized for growth by
304
various Lactobacillus spp. and inhibited the growth of various tested food borne pathogens.
305
In Lactobacillus spp., there were subtle differences in the extent of β-MOS fermentation.
306
Utilization and growth by L. casei var. rhamnosus, L. fermentum and L. plantarum:
307
Feeding studies with DP2 and DP3: Supplementation of MRS media with DP2 and DP3 β-MOS,
308
showed higher growth when compared to respective controls (0.89 to 1.71 log CFU/mL and 1.71
309
to 3.4 fold A600nm when compared to FOS; 0.89 to 1.71 log CFU/mL and 0.03 to 1.35 fold A600nm
310
when compared to glucose and 1.43 to 2.75 log CFU/mL and 5.14 to 23 fold A600nm when
311
compared to media devoid of carbon source). A shift in media pH towards acidic side up to 1.1
312
units was observed in most cultures.
313
Feeding studies with DP5: Supplementation of MRS media with DP5 resulted in lowering of
314
growth parameters (up to 0.74 and up to 6 fold reduction in log CFU/mL and A600nm,
315
respectively) when compared to FOS or glucose. The growth obtained with L. fermentum and L.
316
plantarum was only slightly better (0.46 to 0.69 log CFU/mL and 3.2 fold A600nm) than negative
317
control. Similar to our results, Lactobacillus plantarum NRIC 1547, NRIC 1068 and L. sakei
318
NRIC 0126 showed better growth on oligosaccharides like lactosucrose, 1-kestose and FOS than
14 ACS Paragon Plus Environment
Page 15 of 36
Journal of Agricultural and Food Chemistry
319
glucose.42 A shift in media pH towards acidic side up to 0.8 units was observed in most strains
320
(Table 3).
321
Utilization and growth by L. acidophilus, L. casei, L. brevis and L. helveticus:
322
Feeding studies with DP2 and DP3: Supplementation of MRS media with DP2 and DP3, showed
323
no improvement in log CFU/mL and A600nm when compared to FOS except in L. casei where
324
marginal improvement (0.51 log CFU/mL and 1.1 fold A600nm) in growth parameters was found.
325
Surprisingly, in comparison to glucose, supplementation of MRS media with DP2 and DP3
326
resulted in lower (1.11 to 1.73 fold) A600nm and marginally higher (0.19 to 0.54) log CFU/mL. In
327
comparison to negative control, supplementation of MRS media with DP2 and DP3 resulted in
328
better growth (1.35 to 2.5 log CFU/mL and 3.9 to 8.28 fold A600nm). A shift in media pH towards
329
acidic side (up to 1.1 units) was observed in most cultures.
330
Feeding studies with DP5: Supplementation with DP5 resulted in lowering of growth parameters
331
(0.16 to 1.88 log CFU/mL and 2.6 to 7.2 fold A600nm, respectively) when compared to FOS or
332
glucose. The growth was higher (0.17 to 2.16 log CFU/mL and 1.42 to 2 fold A600nm) only when
333
compared to negative control. A shift in media pH towards acidic side (up to 0.8 units) was
334
observed in most cultures (Table 3).
335
The reasons for better growth response of various tested lactobacilli when fed with DP2
336
and DP3 β-MOS as compared with DP5 β-MOS could be the inherent difference in their
337
carbohydrate fermentation pathways and uptake mechanisms, composition of oligomeric units,
338
and chemical structure along with water solubility.43
339
oligosaccharides of shorter chain length have high water solubility and are quickly fermented,
340
while long chain NDOs are known to be steadily fermented.44 Moura et al.24 found that, L. brevis
341
preferred xylo-oligosaccharides with an average DP of 2 rather than DP 5-6. In other reports,
It has been shown earlier that
15 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 16 of 36
342
mixed mannooligosaccharides from copra meal exhibited prebiotic effect by stimulating the
343
growth of Bifidobacterium infantis and Lactobacillus acidophilus.10
344
Utilization and growth by Lactobacillus spp. on commercial mannobiose (CDP2):
345
Presence of CDP2 (mannopyranose β-1,4 mannopyranose) in MRS media increased the log
346
CFU/mL values (0.02 to 0.36) of L. casei var. rhamnosus, L. fermentum, L. acidophilus and L.
347
brevis while the growth of L. casei, L. plantarum and L. helveticus was found to decrease (0.03
348
to 0.26 log CFU/mL) when compared with glucose. The CDP2 was more preferable (increase of
349
0.03 to 0.36 in log CFU/mL values) than FOS in case of L. casei var. rhamnosus, L. fermentum,
350
L. plantarum and L. helveticus. All the cultures exhibited higher growth (0.88 to 1.9 log
351
CFU/mL) on CDP2 than negative control. A shift in media pH (up to 0.6 units) towards acidic
352
side after fermentation was observed in most cultures.
353
The overall growth obtained on CDP2 was less in various Lactobacillus spp. than ManB-
354
1601 derived DP2 and DP3. The reason for this could be that disaccharides with linkages of 1-2,
355
1-4, and 1-6 are known to generate a high prebiotic index score while mannose containing
356
disaccharides give a low prebiotic index in lactobacilli45 and substituted galactose units in DP2
357
and DP3 could be amenable to α-galactosidases present in Lactobacillus spp.
358
for the substrate promiscuity between ManB-1601 derived DP2, and DP3 in terms of growth in
359
Lactobacillus spp. are difficult to comprehend, as the enzyme systems which are responsible for
360
cleavage of such β-MOS are not known.
361
Utilization and growth by E. coli, L. monocytogenes and S. typhi:
362
The growth of pathogens was lower (up to 0.70 log CFU/mL) in DP2, DP3, and DP5
363
supplemented TSB media when compared with glucose. However, the extent of pathogen
364
inhibition by β-MOS was different as it is known to vary with DP, molecular weight and type of
46, 47
The reasons
16 ACS Paragon Plus Environment
Page 17 of 36
Journal of Agricultural and Food Chemistry
365
functional group present in an oligosaccharide.48 CDP2 supplementation reduced the growth of
366
E. coli and S. typhi, (up to 0.14 log CFU/mL) but marginally promoted L. monocytogenes (0.06
367
log CFU/mL) when compared to glucose. All the food borne pathogens exhibited good
368
correlation between A600nm and log CFU/mL values.
369
Acetate was found as the major SCFA in all the tested probiotic strains and its presence
370
could be one of the reasons for the fall in pH of fermented broth. Our studies on in vitro
371
fermentation of guar gum degradation products by lactobacilli too showed acetate as the chief
372
SCFA component.18 Probiotic bacteria produce SCFAs as an end product of carbohydrate
373
fermentation mediated by glycolytic enzyme and they are known to display several physiological
374
benefits. Acetate in particular has been shown to be beneficial for human muscle, kidney, heart
375
and brain.49 Finally, the following conclusions can be made from in vitro fermentation studies:
376
1) DP3 oligosaccharide supported the growth of all the tested Lactobacillus spp.; 2) DP2
377
oligosaccharide supported the growth of all the tested Lactobacillus spp. except L. helveticus; 3)
378
CDP2 did not support the growth of L. casei, L. plantarum and L. helveticus; 4) DP5 did not
379
support the growth of L. plantarum and L. helveticus, L. casei var. rhamnosus, L. fermentum, L.
380
acidophilus and L. brevis; and, 5) DP2, DP3, and DP5 reduced the growth of E. coli, L.
381
monocytogenes and S. typhi but CDP2 reduced the growth of only E. coli and S. typhi.
382
In conclusion, our studies elucidated that ManB-1601 produced DP2, DP3 and DP5 β-
383
MOS from locust bean gum and belongs to a select group of endo-β-1,4-mannanase which can
384
produce substituted oligosaccharides with galactose at terminal position. In vitro fermentation
385
studies show that the DP2 and DP3 β-MOS were utilised efficiently by various lactobacilli for
386
growth and appear as potential prebiotic candidates when compared to the oligosaccharides (FOS
387
control) available on the market. At present, we are deciphering the hydrolytic action mechanism 17 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 18 of 36
388
of ManB-1601 apart from key determinants responsible for the molecular cross-talk between β-
389
MOS and Lactobacillus spp. with an overall aim to develop potent functional foods based on β-
390
MOS.
391
ACKNOWLEDGEMENTS
392
We thank Prof. Ram Rajashekharan, Director, CSIR-CFTRI, for constant encouragement and
393
support. PKS and DP thank University Grants Commission (UGC), New Delhi, India, and CSIR,
394
New Delhi for providing Junior Research Fellowships. Authors acknowledge the support of
395
MLP 0116 project.
396
REFERENCES
397
1. Chen, M. H.; Swanson, K. S.; Fahey Jr, G. C.; Dien, B. S.; Beloshapka, A. N.; Bauer, L.
398
L.; Rausch, K. D.; Tumbleson, M. E.; Singh, V. In vitro fermentation of
399
xylooligosaccharides
400
microbiota. J. Agric. Food Chem. 2015, 64, 262-267.
produced
from
Miscanthus×
giganteus
by
human
fecal
401
2. Gómez, B.; Gullón, B.; Remoroza, C.; Schols, H. A.; Parajó, J. C.; Alonso, J. L.
402
Purification, characterization, and prebiotic properties of pectic oligosaccharides from
403
orange peel wastes. J. Agric. Food Chem. 2014, 62, 9769-9782.
404
3. Gómez, B.; Míguez, B.; Veiga, A.; Parajó, J. C.; Alonso, J. L. Production, purification,
405
and in vitro evaluation of the prebiotic potential of arabinoxylooligosaccharides from
406
brewer’s spent grain. J. Agric. Food Chem. 2015, 63, 8429-8438.
407
4. Hooper, L. V.; Midtvedt, T.; Gordon, J. I. How host-microbial interactions shape the
408
nutrient environment of the mammalian intestine. Annu. Rev. Nutr. 2002, 22, 283–307.
409
5. In Food Oligosaccharides: Production, Analysis and Bioactivity. Moreno, F. J.; Sanz, M.
410
L. Eds.; John Wiley and Sons: Hoboken, New Jersey, 2014.
18 ACS Paragon Plus Environment
Page 19 of 36
411 412
Journal of Agricultural and Food Chemistry
6. Mussatto, S. I.; Mancilha, I. M. Non-digestible oligosaccharides: a review. Carbohydr. Polym. 2007, 68, 587-597.
413
7. Pedreschi, R.; Campos, D.; Noratto, G.; Chirinos, R.; Cisneros-Zevallos, L. Andean
414
yacon root (Smallanthus sonchifolius Poepp. Endl) fructooligosaccharides as a potential
415
novel source of prebiotics. J. Agric. Food Chem. 2003, 51, 5278-5284.
416
8. Wang, J.; Sun, B.; Cao, Y.; Wang, C. In vitro fermentation of xylooligosaccharides from
417
wheat bran insoluble dietary fiber by Bifidobacteria. Carbohydr. Polym. 2010, 82, 419–
418
423.
419
9. Cuskin, F.; Lowe, E. C.; Temple, M. J.; Zhu, Y.; Cameron, E. A.; Pudlo, N. A.; ...&
420
Gilbert, H. J. Human gut bacteroidetes can utilize yeast mannan through a selfish
421
mechanism. Nature. 2015, 517, 165-169.
422
10. Ghosh, A.; Verma, A. K.; Tingirikari, J. R.; Shukla, R.; Goyal, A. Recovery and
423
purification of oligosaccharides from copra meal by recombinant endo-β-mannanase and
424
deciphering molecular mechanism involved and its role as potent therapeutic agent. Mol.
425
Biotechnol. 2015, 57, 111–127.
426
11. Asano, I.; Hamaguchi, K.; Fujii, S.; Iino, H. In vitro digestibility and fermentation of
427
mannooligosaccharides from coffee mannan. Food Sci. Technol. Res. 2003, 9, 62-66.
428
12. Harjunpää, V.; Teleman, A.; Siika-Aho, M.; Drakenberg, T. Kinetic and stereochemical
429
studies of manno-oligosaccharide hydrolysis catalysed by beta-mannanases from
430
Trichoderma reesei. Eur. J. Biochem. 1995, 234, 278–283.
431
13. McCleary, B. V.; Nurthen, E.; Taravel, F. R.; Joseleau, J. P. Characterisation of the
432
oligosaccharides produced on hydrolysis of galactomannan with β-D-mannase.
433
Carbohydr. Res. 1983, 118, 91–109.
19 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 20 of 36
434
14. von Freiesleben, P.; Spodsberg, N.; Blicher, T. H.; Anderson, L.; Jørgensen, H.;
435
Stålbrand, H.; Meyer A. S.; Krogh, K. B. An Aspergillus nidulans GH26 endo-β-
436
mannanase
437
galactomannans. Enzyme Microb. Technol. 2016, 83, 68-77.
with
a
novel
degradation
pattern
on
highly
substituted
438
15. Chauhan, P. S.; Sharma, P.; Puri, N.; Gupta, N. Purification and characterization of an
439
alkali-thermostable β-mannanase from Bacillus nealsonii PN-11 and its application in
440
mannooligosaccharides preparation having prebiotic potential. Eur. Food Res. Technol.
441
2014, 238, 927–936.
442
16. Chiyanzu, I.; Brienzo, M.; García-Aparicio, M. P.; Görgens, J. F. Application of endo-β-
443
1, 4, D-mannanase and cellulase for the release of mannooligosaccharides from steam-
444
pretreated spent coffee ground. Appl. Biochem. Biotechnol. 2014, 172, 3538-3557.
445
17. Mikkelson, A.; Maaheimo, H.; Hakala, T. K. Hydrolysis of konjac glucomannan by
446
Trichoderma reesei mannanase and endoglucanases Cel7B and Cel5A for the production
447
of glucomannooligosaccharides. Carbohydr. Res. 2013, 372, 60-68.
448
18. Srivastava, P. K.; Kapoor, M. Cost-effective endo-mannanase from Bacillus sp. CFR1601
449
and its application in generation of oligosaccharides from guar gum and as detergent
450
additive. Prep. Biochem. Biotechnol. 2014, 44, 392-417.
451 452 453
19. Yamabhai, M.; Sak-Ubol, S.; Srila, W.; Haltrich, D. Mannan biotechnology: from biofuels to health. Crit. Rev. Biotechnol. 2016, 36, 32-42. 20. Srivastava, P. K.; Kapoor, M. Metal-dependent thermal stability of recombinant endo-
454
mannanase
(ManB-1601)
belonging
to
family
455
CFR1601. Enzyme Microb. Technol. 2016, 84, 41-49.
GH
26
from
Bacillus
sp.
20 ACS Paragon Plus Environment
Page 21 of 36
Journal of Agricultural and Food Chemistry
456
21. Srivastava, P. K.; Kapoor, M. Recombinant GH-26 endo-mannanase from Bacillus sp.
457
CFR1601: Biochemical characterization and application in preparation of partially
458
hydrolysed guar gum. LWT--Food Sci. Technol. 2015, 64, 809-816.
459 460 461 462
22. Miller, G. L. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 1959, 31, 426–428. 23. Dubois, M.; Gilles, K. A.; Hamilton, J. K.; Rebers, P.; Smith, F. Colorimetric method for determination of sugars and related substances. Anal. Chem. 1956, 28, 350-356.
463
24. Moura, P.; Barata, R.; Carvalheiro, F.; Gírio, F.; Loureiro-Dias, M. C.; Esteves, M. P. In-
464
vitro fermentation of xylo-oligosaccharides from corn cobs autohydrolysis by
465
Bifidobacterium and Lactobacillus strains. LWT--Food Sci. Technol. 2007, 40, 963–972.
466
25. Li, H.; Bendiak, B.; Siems, W. F.; Gang, D. R.; Hill Jr, H. H. Carbohydrate structure
467
characterization by tandem ion mobility mass spectrometry (IMMS) 2. Anal. Chem.
468
2013, 85, 2760-2769.
469 470 471 472
26. Biely, P.; Vršanská, M.; Tenkanen, M.; Kluepfel, D. Endo-β-1, 4-xylanase families: differences in catalytic properties. J. Biotechnol. 1997, 57, 151-166. 27. Emi, S.; Fukumoto, J.; Yamamoto, T. Crystallization and some properties of mannanase. Agric. Biol. Chem. 1972, 36, 991-1001.
473
28. McCleary, B. V.; Matheson, N. K. Action patterns and substrate-binding requirements of
474
β-D-mannanase with mannosaccharides and mannan-type polysaccharides. Carbohydr.
475
Res. 1983, 119, 191-219.
476
29. Park, G. G.; Chang, H. G. Separation and preparation of galactosylmanno-
477
oligosaccharides
from
copra
galactomannan
by
mannanase
478
purpurogenum. J. Microbiol. Biotechnol. 1992, 2, 204-208.
from
Penicillium
21 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 22 of 36
479
30. Cerqueira, M. A.; Souza, B. W. S.; Simões, J.; Teixeira, J. A.; Domingues, M. R. M.;
480
Coimbra, M. A.; Vicente, A. A. Structural and thermal characterization of
481
galactomannans from non-conventional sources. Carbohydr. Polym. 2011, 83, 179–185.
482
31. Blibech, M.; Chaari, F.; Bhiri, F.; Dammak, I.; Ghorbel, R. E.; Chaabouni, S. E.
483
Production of manno-oligosaccharides from locust bean gum using immobilized
484
Penicillium occitanis mannanase. J. Mol. Catal. B: Enzym. 2011, 73, 111–115.
485
32. Prado, B. M.; Kim, S.; Özen, B. F.; Mauer, L. J. Differentiation of carbohydrate gums
486
and mixtures using Fourier transform infrared spectroscopy and chemometrics. J.
487
Agric. Food Chem. 2005, 53, 2823-2829.
488
33. Figueiro, S. D.; Góes, J. C.; Moreira, R. A.; Sombra, A. S. B. On the physico-chemical
489
and dielectric properties of glutaraldehyde crosslinked galactomannan–collagen
490
films. Carbohydr. Polym. 2004, 56, 313-320.
491
34. Prashanth, M. S.; Parvathy, K. S.; Susheelamma, N. S.; Prashanth, K. H.; Tharanathan, R.
492
N.; Cha, A.; Anilkumar, G. Galactomannan esters—A simple, cost-effective method of
493
preparation and characterization. Food Hydrocolloids. 2006, 20, 1198-1205.
494
35. Cunha, P. L.; Castro, R. R.; Rocha, F. A.; de Paula, R. C.; Feitosa, J. P. Low viscosity
495
hydrogel of guar gum: Preparation and physicochemical characterization. Int. J. Biol.
496
Macromol. 2005, 37, 99-104.
497
36. Jiang, W.; Zhou, Z.; Wang, D.; Zhou, X.; Tao, R.; Yang, Y.; Shi, Y.; Zhang, G.; Wang,
498
D.; Zhou, Z. Transglutaminase catalyzed hydrolyzed wheat gliadin grafted with chitosan
499
oligosaccharide and its characterization. Carbohydr. Polym. 2016, 153, 105-114.
22 ACS Paragon Plus Environment
Page 23 of 36
Journal of Agricultural and Food Chemistry
500
37. Yakimets, I.; Paes, S. S.; Wellner, N.; Smith, A. C.; Wilson, R. H.; Mitchell, J. R. Effect
501
of water content on the structural reorganization and elastic properties of biopolymer
502
films: a comparative study. Biomacromolecules. 2007, 8, 1710-1722.
503
38. Botosoa, E. P.; Chèné, C.; Blecker, C.; Karoui, R. Nuclear magnetic resonance,
504
thermogravimetric and differential scanning calorimetry for monitoring changes of
505
sponge cakes during storage at 20° C and 65% relative humidity. Food Bioprocess
506
Technol. 2015, 8, 1020-1031.
507
39. Kittur, F. S.; Harish Prashanth, K. V.; Udaya Sankar, K.; Tharanathan, R. N.
508
Characterization of chitin, chitosan and their carboxymethyl derivatives by differential
509
scanning calorimetry. Carbohydr. Polym. 2002, 49, 185–193.
510
40. Moreira, A. S.; Coimbra, M. A.; Nunes, F. M.; Simoes, J.; Domingues, M. R. M.
511
Evaluation of the effect of roasting on the structure of coffee galactomannans using
512
model oligosaccharides. J. Agric. Food Chem. 2011, 59, 10078-10087.
513
41. Tenkanen, M.; Makkonen, M.; Perttula, M.; Viikari, L.; Teleman, A. Action of
514
Trichoderma reesei mannanase on galactoglucomannan in pine kraft pulp. J. Biotechnol.
515
1997, 57, 191-204.
516
42. Endo, A.; Nakamura, S.; Konishi, K.; Nakagawa, J.; Tochio, T. Variations in prebiotic
517
oligosaccharide fermentation by intestinal lactic acid bacteria. Int. J. Food Sci.
518
Nutr. 2016, 67, 125-132.
519
43. Watson, D.; O'Connell Motherway, M.; Schoterman, M. H. C.; Neerven, R. J.; Nauta, A.;
520
Sinderen, V. D. Selective carbohydrate utilization by Lactobacilli and Bifidobacteria. J.
521
Appl. Microbiol. 2013, 114, 1132-1146.
23 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 24 of 36
522
44. Van Laere, K. M.; Hartemink, R.; Bosveld, M.; Schols, H. A.; Voragen, A. G.
523
Fermentation of plant cell wall derived polysaccharides and their corresponding
524
oligosaccharides by intestinal bacteria. J. Agric. Food Chem.2000, 48, 1644-1652.
525 526
45. Sanz, M. L.; Gibson, G. R.; Rastall, R. A. Influence of disaccharide structure on prebiotic selectivity in vitro. J. Agric. Food Chem. 2005, 53, 5192-5199.
527
46. Fredslund, F.; Hachem, M. A.; Larsen, R. J.; Sørensen, P. G.; Coutinho, P. M.; Leggio, L.
528
L.; Svensson, B. Crystal structure of α-galactosidase from Lactobacillus acidophilus
529
NCFM: insight into tetramer formation and substrate binding. J. Mol. Biol. 2011, 412,
530
466-480.
531
47. Carrera-Silva, E. A.; Silvestroni, A.; LeBlanc, J. G.; Piard, J. C.; de Giori, G. S.; Sesma,
532
F. A thermostable α-galactosidase from Lactobacillus fermentum CRL722: genetic
533
characterization and main properties. Curr. Microbiol. 2006, 53, 374-378.
534 535
48. Papineau, A. M.; Hoover, D. G.; Knorr, D.; Farkas, D. F. Antimicrobial effect of water‐soluble chitosans with high hydrostatic pressure. Food Biotechnol. 1991, 5, 45-57.
536
49. Cummings, J. H.; Pomare, E. W.; Branch, W. J.; Naylor, C. P.; Macfarlane, G. T. Short
537
chain fatty acids in human large intestine, portal, hepatic and venous blood. Gut, 1987,
538
28, 1221-1227.
24 ACS Paragon Plus Environment
Page 25 of 36
Journal of Agricultural and Food Chemistry
FIGURE CAPTIONS Figure 1
(A) Biogel P-2 profile of β-MOS generated from locust bean gum by ManB-1601. P1, P2, P3, P4 and P5 represents the β-MOS eluted at fraction no. 31, 39, 49, 57 and 65, respectively. (B) ESI-MS of P1, P2, P3, P4 and P5 fractions obtained after Biogel P-2 chromatography of crude β-MOS derived from locust bean gum after ManB1601 hydrolysis.
Figure 2
FTIR spectra of β-MOS in the spectral region 700-1500 cm-1. (A) DP2, (B) DP3 and (C) DP5.
Figure 3
(A) and (B). XRD analysis of β-MOS DP2 and DP3, respectively. (C) and (D): TGA and DTA curves, respectively, of β-MOS DP2 and DP3,obtained by heating from room temperature at 10 °C/min under an N2 atmosphere.
Figure 4
NMR spectra of Biogel P-2 purified P4 fraction: A) 1H NMR B) 13C NMR
Figure 5
NMR spectra of Biogel P-2 purified P3 fraction: A) 1H NMR B) 13C NMR
Figure 6
Representation of the depolymerization pattern for locust bean gum by purified ManB-1601: Hollow arrows represent cleavage site near substituted region. Solid arrow represents cleavage site near unsubstituted region. Structure 1: Locust bean gum; structure 2: α-D-galactosyl-β-D-mannobiose; structure 3: α-1,6-galactosylmannopyranose; structure 4: mannopyranose β-1,4-mannopyranose; structure 5: Dmannose.
25 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 26 of 36
Table 1 Chemical Shift Values Obtained from 1H NMR for Anomeric Protons for Mannobiose, α-1,6-Galactosyl-mannopyranose and α-D-Galactosyl-β-D-mannobiose
Type of unit
Chemical shift values (ppm) H1α H1β
H1
H1
H1
(non-
Gal
(man-
reducing)
branched)
Mannopyranose β-1,4 mannopyranose4
5.29 4.79
4.66
NA
NA
NA
NA
NA
5.06
4.62
NA
4.76
4.62
5.03
4.58
α-1,6-galactosyl-mannopyranose3 α-D-galactosyl-β-D-mannobiose2 NA: Not assigned; Superscripted numbers represent structure code as mentioned in figure 6
26 ACS Paragon Plus Environment
Page 27 of 36
Journal of Agricultural and Food Chemistry
Table 2 13C NMR Chemical Shift Values for Mannobiose, α-1,6-Galactosyl-Mannopyranose and αD-Galactosyl-β-D-Mannobiose
Chemical shift values (ppm)
Type of unit Mannopyranose β-1,4 mannopyranose4 β-D-mannopyranosyl non reducing end residue
C1
C2
C3
C4
C5
C6
103.39 71.42 72.29 76.28 73.73
59.86
91.89
70.78 72.19 81.08 74.51
62.07
β-D-mannopyranose residue, branched at O-6
93.55
70.25 72.54 76.59 76.13
66.43
α-D-galactopyranosyl residue
99.89
68.68 70.78 69.94
β-D-mannopyranosyl non reducing end residue
99.93
71.21 71.39 76.35 74.74
NA
β-D-mannopyranose residue, branched at O-6
93.55
70.21 72.50 76.52 76.14
66.41
α-D-galactopyranosyl residue
99.84
68.65 70.63 69.96
β-D-mannopyranose reducing end residue α-1,6-Galactosyl-mannopyranose3
NA
60.74/60.25
α-D-Galactosyl-β-D-mannobiose2
NA
60.72/60.24
NA: Not assigned; Superscripted numbers represent structure code as mentioned in figure 6
27 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 28 of 36
Table 3 Growth Characteristics of Lactobacillus spp. and Food Borne Pathogens in Media Containing Purified β-Mannooligosaccharides. β-MOS (0.1% w/v)
Absorbance Log CFU/mL (A600nm) Lactobacillus casei var. rhamnosus DP2 0.73 ± 0.07a 8.0 DP3 0.72 ± 0.07a 7.73 6.3 DP5 0.12 ± 0.0b CDP2 0.61 ± 0.05a 7.2 c Positive control-1 0.42 ± 0.03 6.84 6.84 Positive control-2 0.69 ± 0.09a d Negative control 0.14 ± 0.0 6.3 Lactobacillus casei (NCIM 2126) DP2 0.58 ± 0.05a 7.15 a DP3 0.60 ± 0.06 7.5 DP5 0.10 ± 0.01b 7.16 CDP2 0.62 ± 0.06a 6.7 Positive control-1 0.54 ± 0.04a 6.99 Positive control-2 0.67 ± 0.08a 6.96 5 Negative control 0.07 ± 0.01c Lactobacillus fermentum (NCIM 2165) DP2 0.73 ± 0.06a 8.75 DP3 0.74 ± 0.07a 8.69 b DP5 0.13 ± 0.01 6.69 7.07 CDP2 0.66 ± 0.05a Positive control-1 0.40 ± 0.03c 7.04 a Positive control-2 0.79 ± 0.09 7.04 Negative control 0.04 ± 0.01d 6 Lactobacillus plantarum (NCIM 2372) DP2 0.80 ± 0.01a 7.95 DP3 0.92 ± 0.09a 8.37 DP5 0.13 ± 0.07b 6.3 a CDP2 0.68 ± 0.05 6.99
Final pH
6.0 ± 0.1 6.2 ± 0.1 6.5 ± 0.1 6.5 ± 0.2 6.3 ± 0.1 6.1 ± 0.2 6.5 ± 0.1 6.2 ± 0.2 6.2 ± 0.1 6.4 ± 0.1 6.7 ± 0.1 6.4 ± 0.1 5.9 ± 0.1 6.5 ± 0.1 6.0 ± 0.2 6.0 ± 0.1 6.5 ± 0.2 6.7±0.2 6.4 ± 0.1 5.8±0.2 6.8 ± 0.1 6.0 ± 0.2 6.0 ± 0.1 6.3 ± 0.1 6.7 ± 0.2
MOS (0.1% w/v)
Absorbance Log CFU/mL (A600nm) Lactobacillus acidophilus (NCIM 2285) DP2 0.43 ± 0.04a 7.24 DP3 0.50 ± 0.05a 7.43 b DP5 0.10 ± 0.01 5.47 CDP2 0.67 ± 0.04a 7.2 a Positive control-1 0.53 ± 0.05 7.35 Positive control-2 0.72 ± 0.05c 6.99 d Negative control 0.11 ± 0.01 5.3 Lactobacillus brevis (NCIM 2090) DP2 0.45 ± 0.04a 7.45 a DP3 0.48 ± 0.05 7.45 DP5 0.14 ± 0.01b 6.6 c CDP2 0.65 ± 0.06 6.98 Positive control-1 0.53 ± 0.05a 7.39 d Positive control-2 0.70 ± 0.03 6.96 Negative control 0.09 ± 0.01e 6.1 Lactobacillus helveticus (NCIM 2126) DP2 0.56 ± 0.06a 7.2 DP3 0.64 ± 0.06a 7.31 DP5 0.20 ± 0.02b 7.14 CDP2 0.85 ± 0.08c 7.47 Positive control-1 0.52 ± 0.05a 7.3 d Positive control-2 0.97 ± 0.07 7.5 Negative control 0.10 ± 0.01e 5.78 Listeria monocytogenes (ATCC 13932) DP2 0.7 ± 0.05a 7.73 DP3 0.5 ± 0.07b 7.97 DP5 0.3 ± 0.04c 7.39 CDP2 0.9 ± 0.06d 8.15
Final pH
6.0 ± 0.2 6.1 ± 0.1 6.3 ± 0.1 6.7 ± 0.2 6.3 ± 0.2 5.7 ± 0.2 6.5 ± 0.1 6.2 ± 0.2 6.2 ± 0.2 6.4 ± 0.1 6.6 ± 0.1 6.4 ± 0.1 5.6 ± 0.1 6.5 ± 0.1 6.2 ± 0.2 6.2 ± 0.2 6.3 ± 0.1 6.7±0.1 6.3 ± 0.2 5.7±0.1 6.5 ± 0.1 7.4 ± 0.2 7.6 ± 0.2 8.1 ± 0.1 6.5 ± 0.2 28
ACS Paragon Plus Environment
Page 29 of 36
Journal of Agricultural and Food Chemistry
Positive control-1 0.27 ± 0.01c 6.78 6.0 ± 0.2 Positive control-1 0.2 ± 0.03e 7.18 8.5 ± 0.2 a f Positive control-2 0.68 ± 0.08 7.04 6.2 ± 0.1 Positive control-2 0.9 ± 0.1 8.09 7.8 ± 0.2 Negative control 0.04 ± 0.01d 5.84 6.5 ± 0.1 Negative control 0.3 ± 0.04g 7.41 7.0 ± 0.1 Escherichia coli (ATCC 11775) Salmonella typhi (ATCC 25241) DP2 2.8 ± 0.05a 8.3 8.2 ± 0.2 DP2 2.0 ± 0.12a 7.72 8.3 ± 0.1 a a DP3 2.8 ± 0.1 8.6 8.5 ± 0.2 DP3 2.1 ± 0.08 7.64 8.3 ± 0.2 DP5 3.0 ± 0.1b 8.62 7.9 ± 0.2 DP5 2.1 ± 0.09a 7.59 8.7 ± 0.2 a a CDP2 2.8 ± 0.08 8.68 8.5 ± 0.1 CDP2 2.1 ± 0.13 7.68 8.3 ± 0.1 8.56 8.6 ± 0.2 Positive control-1 2.0 ± 0.1a 7.72 8.4 ± 0.1 Positive control-1 2.7 ± 0.02a Positive control-2 3.7 ± 0.1c 8.79 8.3 ± 0.2 Positive control-2 3.1 ± 0.13b 7.82 8.2 ± 0.1 8.49 8.5 ± 0.1 Negative control 2.0 ± 0.05a 7.58 8.5 ± 0.1 Negative control 2.7 ± 0.1a Positive control-1- MRS media containing fructo-oligosaccharides; Positive control-2: Glucose (0.1%w/v); Negative control- MRS media devoid of carbon source; Absorbance values of each lactobacilli sp. indicated with different superscripted letters (a, b, c, d, e, f, g) are statistically different (significant at 0.01 level), while values indicated with same superscripted letters are not statistically different (significant at 0.01 level); DP2: mannopyranose β-1,4 mannopyranose4 or α-1,6-galactosyl-mannopyranose3; DP3: α-Dgalactosyl-β-D-mannobiose2 (superscripted numbers represent structure code as mentioned in figure 6).
29 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 30 of 36
Figure 1
A 2.5 P1
A480nm
2.0
P2
P3
P4 P5
1.5 1.0 0.5 0.0 0
20
40
TOF MS ES+ 243
49
22081402 19 (0.368) Cm (12:20) 850.6722 100
TOF MS ES+ 22081403 21 (0.407) Cm (20:23) 527.3005 867 100
P2
TOF MS ES+ 1.61e3
P3
%
P1
100
%
22081401 88 (1.681) Cm (84:91) 599.1530 100
80
Fraction no. B
39
31
60
% 599.7004
845.7578 851.7042
1173.9666
596.7271 680.0605
1030.3115
0
m/z 600
800
1000
1200
1400
1600
1800
200
400
600
800
1000
1200
1400
1600
1800
0
m/z 200
400
600
800
1000
1200
1400
1600
1800
65
57 22081404 41 (0.787) Cm (35:44) 365.5380 100
TOF MS ES+ 1.89e3
P4
22081405 46 (0.882) Cm (43:50) 100
%
400
m/z
203.6782
TOF MS ES+ 85
P5
%
200
0
366.5493
707.0107
381.5049
0
m/z 200
400
600
800
1000
1200
1400
1600
1800
223.7091 365.5768 605.4595
0
ACS Paragon Plus Environment
30
Page 31 of 36
Journal of Agricultural and Food Chemistry
Figure 2
31 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 32 of 36
Figure 3
50
50
A
30
20
10
30
20
10
0
0
0
10
20
30
40
50
2θ
60
70
0
80
10
20
30
40
50
60
70
80
2θ
120
0
C Heat flow (mW/mg)
80
DP2
60
D
-5
100
Weight (%)
B
40
Intensity (coulumbs)
Intensity (coulumbs)
40
40
-10
171 318 516
-20
DP2
150 -25
314
DP3
20
DP3
-15
-30
510 0
-35 0
200
400
600
800
0
200
0
400
600
800
0
Temperature ( C)
Temperature ( C)
32 ACS Paragon Plus Environment
73
72
71
70
69 92
68
67 90
66 88
65 3.50
86
64 3.45
Mannobiose
84
63
ACS Paragon Plus Environment
2.52
4.2
3.40
82
4.1
3.35
80
62
61
4.0
3.30
78 76
60
59 73.738
3.55
4.3
74.517
4.4
76.590 76.283 76.134
4.5
81.085
3.60
4.68
2.05
1.22
0.54
4.6
59.860
94 3.65
3.41
3.70 2.59
2.08
3.75 4.7
4.622
4.663
4.791
5.060 5.057
5.292 5.284
3.951 3.945 3.926
4.101 4.083
Mannobiose
60.258
96 4.8
61.111 60.747
98 4.9
62.070
100 3.80
91.889
93.553 93.417
3.85 3.12
3.90 5.0
66.439
102 5.1
68.957 68.680
2.00
5.2
4.91
4.36
3.95
70.787 70.621 70.351 70.255 69.940
99.942 99.896
5.3
71.429
B 2.52
3.951 3.945 3.926 3.909 3.878 3.872 3.868 3.850 3.844 3.840 3.819 3.815 3.781 3.771 3.767 3.746 3.742 3.732 3.725 3.710 3.707 3.700 3.692 3.689 3.666 3.657 3.640 3.633 3.629 3.619 3.615 3.608 3.605 3.595 3.548 3.533 3.529 3.527 3.523 3.469 3.467 3.463 3.447 3.444 3.438 3.428 3.425 3.417 3.381 3.363 3.344 3.339 3.334 3.325 3.320 3.315 3.305 3.301
A
72.542 72.296 72.124
103.398
Page 33 of 36 Journal of Agricultural and Food Chemistry
Figure 4
ppm
ppm
ppm
ppm
33
75 98
70 96 94
65
3.40
92
3.35
90
60
3.30
88
3.25 3.20
86
ppm
84
55
ACS Paragon Plus Environment 2.70
82
50
2.65
80
2.60 2.55 ppm
0.39
1.68
3.8 1.12
3.61
3.9
2.568
4.0 3.82
4.1
2.615 2.600
4.2
1.03
4.620 4.616 4.589
4.766
5.033 5.031
3.987 3.984 3.923 3.917 3.898 3.853 3.846 3.828 3.818 3.814 3.789 3.762 3.747 3.742 3.732
4.068
Triose
1.77
1.66
4.3
0.33
3.45
4.4
0.30
4.5
1.85
3.50 0.77
4.6
0.98
3.67
3.55 0.51
4.7
Triose Mannotriose 76.520 76.359 76.205 76.141
3.60
2.00
4.8
43.068
93.558 93.413
0.15
1.00
4.9
60.727 60.243
100
3.65
4.54
3.70
1.35
5.00
1.12
5.0
66.414
B 99.934 99.841
3.747 3.742 3.732 3.727 3.721 3.717 3.707 3.684 3.676 3.670 3.653 3.648 3.641 3.633 3.622 3.618 3.609 3.602 3.597 3.588 3.577 3.563 3.523 3.517 3.504 3.498 3.435 3.415 3.405 3.396 3.378 3.368 3.361 3.356 3.343 3.313 3.309 3.299 3.294 3.290 3.280 3.276
A
72.501 71.396 71.215 70.639 70.384 70.213 69.968 69.688 69.157 68.655
74.747 74.516
Journal of Agricultural and Food Chemistry Page 34 of 36
Figure 5
ppm
78 ppm
45
ppm
34
Page 35 of 36
Journal of Agricultural and Food Chemistry
Figure 6
35 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 36 of 36
Table of Contents Graphic
36 ACS Paragon Plus Environment