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Sweetness and Sensory Properties of Commercial and Novel Oligosaccharides Of 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.
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