1 Sweetness and sensory properties of commercial and novel oligosaccharides of
2 prebiotic potential
3
4 Laura Ruiz-Aceitunoa, Oswaldo Hernandez-Hernandeza, Sofia Kolidab, F. Javier
5 Morenoa,* and Lisa Methvenc
6
7 a Institute of Food Science Research, CIAL (CSIC-UAM), Nicolás Cabrera 9, 28049
8 Madrid (Spain)
9 b OptiBiotix Health plc, Innovation Centre, Innovation Way, Heslington, York YO10
10 5DG (UK)
11 c Sensory Science Centre, Department of Food and Nutritional Sciences, The University
12 of Reading, PO Box 226, Whiteknights, Reading RG6 6AP (UK)
13
14 *Corresponding author: [email protected] Tel (+34) 91 0017948
1
15 Abstract
16 This study investigates the sweetness properties and other sensory attributes of ten
17 commercial and four novel prebiotics (4-galactosyl-kojibiose, lactulosucrose, lactosyl-
18 oligofructosides and raffinosyl-oligofructosides) of high degree of purity and assesses the
19 influence of their chemical structure features on sweetness. The impact of the type of
20 glycosidic linkage by testing four sucrose isomers, as well as the monomer composition
21 and degree of polymerization on sweetness properties were determined. Data from the
22 sensory panel combined with principal component analysis (PCA) concludes that chain
23 length was the most relevant factor in determining the sweetness potential of a
24 carbohydrate. Thus, disaccharides had higher sweetness values than trisaccharides which,
25 in turn, exhibited superior sweetness than mixtures of oligosaccharides having DP above
26 3. Furthermore, a weak non-significant trend indicated that the presence of a ketose sugar
27 moiety led to higher sweetness. The novel prebiotics tested in this study had between 18
28 and 25% of relative sweetness, in line with other commercial prebiotics, and samples
29 varied in their extent of off flavour. Therefore, these findings suggest a potential use for
30 clean tasting prebiotics as partial sugar replacers, or in combination with high intensity
31 sweeteners, to provide a well-balanced sweetness profile.
32
33 Keywords: sweetener; enzymatic synthesis; sensory evaluation; free sugar substitute;
34 non-digestible oligosaccharides.
2
35 1. Introduction
36 A high level of free sugars intake is associated with poor dietary quality, dental caries,
37 obesity or diabetes among other noncommunicable diseases (WHO/FAO Expert
38 Consultation, 2003). Free sugars are defined as monosaccharides and disaccharides added
39 to foods and beverages and sugars naturally present in honey, syrups, fruit juices and fruit
40 juice concentrates. In 2015, the World Health Organization (2015) published a guideline
41 on sugar intake for adults and children where the main and strong recommendation was
42 to reduce the intake of free sugars to less than 10% of total energy intake, with a
43 conditional recommendation for further reduction to below 5% of total energy intake.
44 Different policy-makers have rapidly taken into account these recommendations and
45 some governments have introduced tax on sugary drinks, among other measures
46 developed to decrease the intake of free sugars (Briggs, 2016). In this scenario, it has been
47 recently reported that the reformulation to reduce sugar concentration in sweetened
48 beverages could be the most beneficial and healthy industry strategy (Briggs et al., 2017).
49 Therefore, the use of high-potency sweeteners (also known as non-nutritive sweeteners
50 or low-calorie sweeteners) and/or their blending with sugars is recognized as a
51 technologically feasible, economically viable and effective strategy in reducing free
52 sugars in foodstuffs (Gibson et al., 2017a; Di Monaco, Miele, Cabisidan, & Cavella,
53 2018). The current high-intensity sweeteners (HIS) more commonly used in Europe are
54 synthetic, such as aspartame, saccharin, sucralose, acesulfame-K, neotame, although
55 some of them are derived from a natural source as is the case of steviol glycosides.
56 However, due to the absence of solid scientific evidence supporting the role of synthetic
57 sweeteners in preventing weight gain, together with the lack of studies on other long-term
58 effects on health, the use of common synthetic sweeteners as part of a healthy diet is
59 currently under question (Edwards, Rossi, Corpe, Butterworth, & Ellis, 2016; Borges et
3
60 al., 2017; Azad et al., 2017). In addition, HIS tend to have a different sweetness profile
61 to natural sugars, often having a lingering sweetness and, in some cases, additional off-
62 notes such as bitterness or specific flavours such as liquorice (Prakash, Dubois, Clos,
63 Wilkens, & Fosdick, 2008). In this context, it has been claimed that the replacement of
64 free sugars with any HIS will continue to be primarily governed by the required sweetness
65 profile, making sensory science and in-depth understanding of consumer attitude key
66 players on the potential incorporation of any new sweetener into a normal diet (Miele et
67 al., 2017).
68 Carbohydrates with prebiotic properties, which are selectively utilized by host
69 microorganisms conferring health benefit(s) to the gastrointestinal tract (GIT), among
70 other body sites (Gibson et al., 2017b), exhibit a high resistance to digestion and
71 absorption in the upper GIT having, thus, a low calorific content. Prebiotic carbohydrates
72 are mainly produced either by extraction from natural sources, as well as by enzymatic
73 hydrolysis or synthesis using naturally-occurring polysaccharides or disaccharides (such
74 as lactose, sucrose and maltose) (Díez-Municio, Herrero, Olano, & Moreno, 2014). The
75 assessment of the sweetness properties of oligosaccharides with prebiotic properties, or
76 with slow digestion rate, produced from natural sources and "green technology" can
77 provide valuable insights to better understand their potential as suitable and healthy low-
78 calorie sweeteners. Although there are several studies dealing with the determination of
79 the sweetness of carbohydrates, such as those evaluating monosaccharides (Schaafsma,
80 2002; Gwak, Chung, Kim, & Lim, 2012), maltodextrins (Marchal, Beeftink, & Tramper,
81 1999; Pullicin, Penner, & Lim, 2017), lactose (Pangborn & Gee, 1961), glucose polymers
82 (Lapis, Penner, & Lim, 2014) or polyalcohols (Gwak et al., 2012; Grembecka, 2015), the
83 information gathered on commercial prebiotic carbohydrates, such as lactulose, FOS,
84 GOS or XOS, is scarce (Parrish, Talley, Ross, Clark, & Phillips, 1979; Niness, 1999;
4
85 Schaafsma, 2008; Bali, Panesar, Bera & Panesar, 2015; Samanta et al., 2015).
86 Interestingly, recent works have demonstrated that the incorporation of GOS (Belsito et
87 al., 2017) or XOS (Ferrao et al., 2018) into processed cheese led to an improvement of
88 the sensory characteristics.
89 In recent years, the effective production of a series of novel prebiotic oligosaccharides
90 enzymatically synthesized, using microbial transglycosidases acting on sucrose, has been
91 reported (Diez-Municio, Kolida, Herrero, Rastall, & Moreno, 2016a), and whose
92 sweetness potential is unknown. Thus, the objective of this work is to comparatively
93 evaluate the sweetness and flavour profiles of fourteen different carbohydrates, including
94 novel prebiotics as well as a range of commercially available carbohydrates in order to
95 infer findings from the relationship between the structural features and the sweetness
96 properties of the tested carbohydrates.
97
98 2. Material and methods
99 2.1. Carbohydrates and chemicals
100 Orafti® HP, Orafti® P95 and Palatinose® were acquired from Beneo-Orafti
101 (Tienen, Belgium) and IMO Syrup (isomaltooligosaccharide) was bought from Vitafiber
102 (Bioneutra, Alberta, Canada). Kojibiose, leucrose, maltulose and turanose were acquired
103 from Carbosynth (Compton, UK). Lactose and lactulose were purchased from Sigma-
104 Aldrich (Steinheim, Germany). All material was stored at ambient temperature, except
105 for IMO Syrup which was stored at 5 °C.
106 Water (Harrogate Spa mineral water) and white granulated sugar (Sainsburys,
107 London, UK) used for sensory testing were purchased in local supermarkets in Reading
108 (UK).
109
5
110 2.2.Synthesis and purification of novel oligosaccharides
111 The novel carbohydrates were produced by enzymatic synthesis using microbial
112 transglycosidases acting on sucrose. 4-Galactosyl kojibiose (β-D-Gal-(1→4)-D-Glc-
113 (2→1)-α-D-Glc) was produced as described by Diez-Municio et al. (2012a),
114 lactulosucrose (-D-Gal-(1→4)--D-Fru-(2→1)--D-Glc) as in Diez-Municio, Herrero,
115 Jimeno, Olano & Moreno (2012b), lactosyl-oligofructosides (LFOS) (β-D-Gal-(1→4)-α-
116 D-Glc-[(1→2)-β-D-Fru]n, n = 2–4) as in Diez-Municio et al. (2015) and raffinosyl-
117 oligofructosides (RFOS) (α-D-Gal-(1→6)-α-D-Glc-[(1→2)-β-D-Fru]n, n = 2–5) as in
118 Diez-Municio et al. (2016b).
119 The synthesized carbohydrates were isolated and purified by high performance
120 liquid chromatography with refractive index detector (HPLC-RID) from the
121 corresponding reaction mixtures on an Agilent Technologies 1260 Infinity LC System
122 (Boeblingen, Germany) using a Zorbax NH2 PrepHT preparative column (250 mm x 21.2
123 mm, 7 µm particle size) (Agilent Technologies, Madrid, Spain). Two mL of reaction
124 mixtures (approx. 150 mg of total carbohydrates) were eluted with acetonitrile:milli-Q®
125 ultrapure water with a resistivity of 18.2 MΩ·cm at 25 °C (75:25, v:v) as the mobile phase
126 at a flow rate of 21 mL/min for 30 min. The separated compounds were collected using
127 an Agilent Technologies 1260 Infinity preparative-scale fraction collector (Boeblingen,
128 Germany) and the fractions were evaporated in a rotatory evaporator R-200 (Büchi,
129 Flawil, Switzerland) at a temperature below 25 ºC and freeze-dried to avoid any cross
130 contamination (microbial or chemical).
131 The obtained purified oligosaccharides were sterilized by filtration (0.22 μm
132 filter). Moreover, in order to ensure all solvent was removed, total carbon, hydrogen,
133 nitrogen and sulfur contents were determined in all the carbohydrates using a LECO
6
134 analyzer (Model CHNS-932, Leco Corp., St Joseph, MI) from the Service
135 Interdepartmental Research (SIdI-UAM) in Madrid.
136 All samples underwent microbiological clearance testing. The presence of yeast
137 and molds, total and sporulated aerobic microorganisms and enterobacteria were analyzed
138 in the samples. Serial dilutions were performed in triplicate with peptone water (Biocult
139 BV, Roelofarendsveen, The Netherlands). Yeast and molds were plated on Sabouraud
140 chloramphenicol agar and incubated at 25 ºC for 5 days. The total and sporulated aerobic
141 bacteria were determined by plating appropriately diluted samples onto plate count agar.
142 The samples were incubated at 30 ºC for 72 h for total aerobic bacteria and at 37 ºC for
143 48 h for sporulated aerobic bacteria after heat treatment of stock dilution at 80 ºC for 10
144 min. For enterobacteria counts, violet red bile dextrose agar was used and incubation was
145 carried out at 30ºC for 24h. All microbial counts were reported as colony forming units
146 per gram (cfu g-1). All culture media were of Difco (Becton, Dickinson & Company,
147 Franklin Lakes, NJ, USA).
148
149 2.3.Conditions for sensory analysis
150 The sweetness intensity of commercial and novel prebiotic oligosaccharides was
151 evaluated using an experienced sensory evaluation panel of subjects. The study was given
152 approval by the University of Reading Research Ethics Committee (UREC study number
153 16_19). Sensory analysis was performed in an air-conditioned (23-24°C, room
154 temperature) sensory laboratory with individual booths and artificial daylight.
155 The sweetness intensity and several flavor attributes of novel and commercial
156 oligosaccharides was carried out by a screened and trained sensory panel which consisted
157 of 10 panelists (9 female, 1 male; 30-60 years of age) with between 5 months and 8 years’
158 experience.
7
159 The panelists were trained at the Sensory Science Centre (Department of Food
160 and Nutritional Sciences, University of Reading, UK). Using a QDA (quantitative
161 descriptive analysis) profiling approach the panel first developed a consensus vocabulary
162 and then scored independently each attribute, in duplicate.
163 The panel used 11 attributes to define the oligosaccharide samples (sweet, overall
164 strength of off taste/flavor, bitter, cardboard/stale, candyfloss, sour, metallic, salty, crusty
165 bread, perfume flavour and sweet aftertaste) as defined in Table 1.
166 The training focused on ensuring each panelist could reliably score sweetness
167 relative to four sucrose standards (5, 10, 20 and 26 g/L). The average panel ratings for
168 these standards were 10, 35, 75 and 100 respectively on a 0-100 line scale, and hence
169 these four positions were used as anchors to provide a structured scale on which to rate
170 all oligosaccharide samples. All other attributes were scored as relative values using
171 unstructured line scales (0-100). Due to the limited sample availability each panelist was
172 presented with only 0.5 ml of sample for each scoring session. Therefore, training
173 additionally focused on ensuring panelists were able to sip this small sample volume from
174 a 30 ml transparent polystyrene cup and allow it to flow over the top of their tongue
175 before swallowing and scoring sweetness reproducible. Palate cleansing before and
176 between sample scoring was done using filtered water and low salt crackers (Carr’s water
177 crackers, United Biscuits Ltd., Hayes, UK).
178 Oligosaccharide samples were prepared as a 50 g/L solution (weighed to an
179 accuracy of ±0.005 g) in mineral water (Harrogate Spa mineral water), stirring over a
180 magnetic plate to ensure thorough sample dispersion. In a pilot tasting session it was first
181 ensured that 50 g/L was of sufficient concentration to be tasted by all panel members
182 (data not shown); a higher concentration was not used due to limited sample availability.
183 The samples dispersed well and solubilized easily in water, with the exception of
8
184 raffinosyl-oligofructosides (RFOS) which were more difficult to disperse and separated
185 out of solution on standing. However, all samples were shaken immediately before
186 serving to each sensory panelist. Samples were labelled with random 3-digit codes and
187 sample order presentation was done in a monadic sequential manner.
188 The sucrose standards were presented at the start of each panel rating session for re-
189 familiarization in order that the panelists could score the sweetness of the
190 oligosaccharides accurately against the standard anchors.
191 The mean sweet ratings of the four sucrose standards were used to plot a dose-
192 response curve, the linear regression for which was Perceived Sweetness = 37.5 x Sucrose
193 Concentration (g/L) (r2 = 0.98). The mean sweet ratings for each 50 g/L oligosaccharide
194 were the converted to equivalent sweetness (ES) values from this equation. Sugars and
195 sweeteners are usually compared to sucrose by relative sweetness (RS) values, the ES on
196 a dry weight basis. To account for the 50 g/L of each oligosaccharide, the RS was
197 determined as RS = ES /50.
198
199 2.4.Statistical analysis.
200 Data were analyzed using a mixed model ANOVA where panelists were treated as
201 random effects and samples as fixed effects, the main effects were tested against the
202 sample by assessor interaction. Multiple pairwise comparisons were carried out using
203 Fishers LSD and a significant difference was declared at an alpha risk of 5% (p 0.05).
204 Data analysis was carried out using Senpaq software (Qi Statistics, Reading, UK).
205 PCA tests and Spearman rank correlation analyses were done using the statistical
206 software XLSTAT (Addinsoft, version 2015, Paris, France).
207
208 3. Results and Discussion
9
209 3.1. Chemical structure and degree of purity of the tested carbohydrates
210 Table 2 shows the chemical structures and degree of purity of the carbohydrates
211 used in the present study. A range of sucrose isomers sharing the monomer composition
212 but differing in the glycosidic bond (leucrose, maltulose, turanose and palatinose) were
213 included in order to determine the potential influence on the glycosidic linkage on the
214 resulting sweetness. In addition, lactose and lactulose were assayed as disaccharides
215 forming the core structure of some of the novel prebiotics tested. Moreover, a wide range
216 of degrees of polymerization (DP) were studied (from 2 to an average of 23). Glycosidic
217 linkages varied in the structures (α(1→2), β(1→4) and β(2→1) bonds), and the
218 monomeric composition was based on glucose, galactose or fructose, which are the main
219 building blocks of the majority of food oligosaccharides presently available or in
220 development as functional food ingredients. For instance, kojibiose consists of two
221 glucose units linked by an α(1→2) bond, whereas 4-galactosyl-kojibiose contains three
222 monomers (two glucose and one galactose units), and lactulosucrose has three different
223 monomers (galactose, fructose and glucose) linked by (1→4) and (2→1) bonds. In the
224 case of RFOS, which contained galactose, glucose and up to five molecules of fructose,
225 are linked by (1→6) and (2→1), respectively. Compounds having a higher DP, such
226 as commercial oligofructoses, isomaltooligosaccharides and long chain inulin were also
227 included.
228 The degree of purity was determined in all assayed carbohydrates in order to avoid
229 any bias in the sweetness properties induced by the possible presence of minor
230 carbohydrates, especially monosaccharides. The levels of purity were satisfactory and
231 ranged from 87 to 99% (Table 2).
232 Microbiological assays showed that the microbial load (yeast and molds, total and
233 sporulated aerobic bacteria, enterobacteria) was, in all assayed carbohydrates, lower than
10
234 103 cfu g-1, indicating that the synthesized oligosaccharides were microbiologically safe
235 and could be used as a food ingredient.
236 Determination of carbon, hydrogen, nitrogen and sulfur in the novel oligosaccharides
237 (i.e., 4-galactosyl-kojibiose, lactulosucrose, RFOS and LFOS) revealed normal values for
238 these elements, including low nitrogen contents (between 3 and 5.7 g/L) which is in
239 accordance with their high degree of purity.
240
241 3.2. Sensory profile of commercial and novel carbohydrates
242 Significant differences in sweet taste were found for the oligosaccharides tested
243 with mean scores ranging from 11.2 to 68.3 (out of 100) (Table 3). Turanose was
244 significantly sweeter than all other samples except leucrose. The sweet scores for
245 disaccharides ranged from 49.8 to 63.0. Kojibiose was the disaccharide with the lowest
246 sweet mean value (49.8), it was significantly less sweet than both leucrose and turanose
247 and was not significantly different from either of the trisaccharides, lactulosucrose (46.5)
248 and 4-galactosyl-kojibiose (41.4). Among the oligosaccharides having a DP above 3, the
249 sweetest samples were the oligofructose with relatively low DP (Orafti® P95), and
250 RFOS, followed by LFOS and IMO syrup, whereas the long chain inulin (Orafti® HP)
251 was noticeably the least sweet sample. Differences in sweet aftertaste (post swallowing)
252 followed the same trend (Table 3). The relative sweetness (RS) of the oligosaccharides
253 varied from 0.06 to 0.36, indicating that on a weight basis these molecules had between
254 6% and 36% the sweetness of sucrose.
255 The differences in the overall strength of off taste/flavours in the oligosaccharide
256 samples were also significant with palatinose having the least off flavor value (8.9), and
257 kojibiose having a significantly higher level than all other assayed carbohydrates.
258 Although bitter taste and cardboard/stale flavor values were particularly low in all
11
259 carbohydrates, values determined for lactosyl-oligofructosides (LFOS) were significantly
260 higher than in the rest of the studied carbohydrates. None of the remaining off-notes
261 characterized were significantly different between samples. There was a candyfloss
262 (cooked sugar) flavor at low levels in some samples, particularly in leucrose and
263 maltulose that was absent in LFOS. Crusty bread flavor tended to be slightly higher in the
264 commercial oligosaccharides (DP≥3), specifically in oligofructose (Orafti P95®), while
265 the novel oligosaccharides did not present this attribute. Kojibiose and 4-galactosyl-
266 kojibiose were rated slightly higher for the perfume note, although at a low level with no
267 significant differences between samples. Lastly, sour (rancid), salty and metallic did not
268 appear to substantially contribute to the overall off flavour, nor to discriminate between
269 samples.
270 In order to better correlate sweetness and chemical structure of the tested
271 carbohydrates, a multivariate analysis was carried out with the aim to group the different
272 carbohydrates and visualize main trends. Concretely, Figure 1 graphically shows the
273 Principal Component Analysis (PCA) of the sweet scores and DP, using two other factors
274 regressed onto the plot as supplementary variables (presence of ketose groups and types
275 of linkage). The main factor contributing to sweetness was a low DP, which is in good
276 agreement with previous findings (Kaulpiboon, Rudeekulthamrong, Watanasatitarpa, Ito
277 & Pongsawasdi, 2015). As can be seen in Figure 1 the DP and mean sweet score are at
278 opposite sides of dimension 1, the Spearman’s correlation coefficient between the two
279 was -0.87 (p<0.0001) as indeed all oligosaccharides with a DP above 3 were substantially
280 less sweet. Moreover, presence of a ketose sugar moiety did not have a significant
281 influence on sweetness although there was a very weak non-significant trend that the
282 presence of a ketose sugar led to higher sweetness (Spearman’s correlation coefficient
283 0.17, p=0.56). This weak trend could partly explain the fact that kojibiose, the only tested
12
284 disaccharide comprised by two glucose monomers, had a lower sweetness than the
285 sucrose isomers or lactulose (comprising galactose and fructose monomers). In this sense,
286 Schaafsma (2002) stated that fructose has higher sweetness properties than glucose.
287 Moreover, despite turanose (α-D-Glc-(1→3)-β-D-Fru) was significantly sweeter than
288 maltulose (α-D-Glc-(1→4)-β-D-Fru) and palatinose (α-D-Glc-(1→6)-β-D-Fru) but not
289 significantly sweeter than leucrose (α-D-Glc-(1→5)-β-D-Fru), PCA revealed the lack of a
290 clear relationship between the type of linkage and sweetness of the resulting
291 oligosaccharide (Figure 1).
292 Concerning the novel prebiotics tested in this study, the purified trisaccharides
293 lactulosucrose and 4-galactosyl-kojibiose had around 25% of the sweetness of sucrose
294 which was similar to or even higher than the relative sweetness of oligofructose with low
295 DP (Orafti® P95) (Table 3) which has previously been described as sweet with a pleasant
296 flavor and, consequently, it could be used in combination with high intensity sweeteners
297 to replace sucrose, providing a well-balanced sweetness profile (Niness, 1999). LFOS
298 (DP 4-6) and RFOS (DP 4-7), which exhibited around 18% of the sweetness of sucrose,
299 were significantly sweeter than the long-chain inulin (Table 3).
300 Finally, the tested oligosaccharides were also characterized by other flavour
301 attributes, however, the results showed no clear association regarding off notes related to
302 commercial or non-commercial oligosaccharides.
303
304 4. Conclusions
305 Information related to sweetness and sensory properties of prebiotic
306 oligosaccharides which could potentially be used as sweeteners is rather scarce at present.
307 The present study may initially contribute to fill this gap because a wide range of
308 commercial and novel prebiotic oligosaccharides displaying different chemical
13
309 structures, such as degree of polymerization, monomer composition and order, presence
310 of ketone vs. aldehyde group, or different glycosidic linkages, were assayed in order to
311 propose relationships between carbohydrate structure and sweetness properties. Data
312 from the sensory panel and further supported by PCA pointed out that chain length was
313 the most relevant factor in determining the sweetness potential of a carbohydrate. Thus,
314 disaccharides had higher sweetness values (49.8-68.3) than trisaccharides (41.4-46.5)
315 which, in turn, exhibited greater sweetness than mixtures of oligosaccharides having DP
316 above 3 (11.2-37.1). Less remarkably, a weak and non-significant trend indicated that the
317 presence of a ketose sugar moiety led to higher sweetness, whereas the type of glycosidic
318 linkage did not have a clear impact on the sweetness properties of the tested
319 oligosaccharides.
320 The novel prebiotic oligosaccharides studied in the current study had between 18
321 and 25% of the sweetness of sucrose (relative sweetness), showing, thus, a sweetness
322 potential in line with other commercial prebiotics. Therefore, these findings suggest a
323 potential use for clean tasting prebiotics as partial sugar replacers, or in combination with
324 high intensity sweeteners, to provide a well-balanced sweetness profile.
325
326 Acknowledgments
327 This work has been funded by Optibiotix Health plc (York, UK) and by Ministerio de
328 Economía, Industria y Competitividad (MINEICO) of Spain (project AGL2017-84614-
329 C2-1-R). L. R-A. thanks the Spanish Research Council (CSIC) and the Spanish Ministry
330 of Economy and Competitiveness for a “Juan de la Cierva-Formación” contract.
331
14
332 Declarations of interest: The authors declare the following competing financial
333 interest(s): SK is the Research and Development director of Optibiotix Health plc.
15
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468 Figure captions
469 Figure 1. Principal component analysis (PCA) plot of the sweet scores and degree of
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471 Additional supplementary variables as presence of ketose groups and type of linkage were
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473 Observations plot.
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