Exopolysaccharide (EPS), emulsion comprising the same and use of the same

Abstract

The present invention relates to a chemical compound of a novel exopolysaccharide, so called Stellan, produced by Sphingomonas sanxanigenens, an emulsion composition with dual texturizing and emulsifying properties containing the compound and its use in several food applications.

Description

[0001]EXOPOLYSACCHARIDE (EPS), EMULSION COMPRISING THE SAME AND USE OF THE SAME FIELD OF THE INVENTION The present invention relates to a new exopolysaccharide produced by Sphingomonas sanxanigenens, an emulsion composition containing the same and its application in food. BACKGROUND OF THE INVENTION Microbial exopolysaccharides have attracted attention from industrial and academic com- munity for their origin as biobased polymers, sustainable production, interesting mono- saccharide conformation, and outstanding physicochemical and functional properties. Generally, an EPS is recognized as a homopolysaccharide or heteropolysaccharide com- posed of only one type of monosaccharides or two or more than two different monosac- charides as a repeating unit, respectively. These may be anionic, neutral, cationic, or zwitterionic and in most cases found in ordered compositions ranging in molar mass from 0.5 to 2.0 x 106 Da. Thus, the monosaccharide composition, type and degree of substi- tution (side groups), molecular weight, and the conformational state of the structure are directly related to the unique properties of EPS. These characteristics are highly related to the functional properties. The present invention describes the chemical structure of a novel exopolysaccharide (EPS); herein so-called Stellan, produced by Sphingomonas sanxanigenens. Earlier pub- lications, such as CN114014944, disclose that Sphingomonas sanxanigenens produces a typical sphingan with chemical structure composed of glucose, mannose, rhamnose, and glucuronic acid. This patent discloses that Stellan, which can be extracted by hot alcohol precipitation, presents a unique chemical structure and surface-active properties. Stellan shows dual functionality such as a stabilizer and an emulsifier at various concentrations and oil ratios. Moreover, the emulsions can present thermo-reversible gel and thixotropic shear reversible behavior. This patent demonstrates that Stellan can replace proteins, emulsifiers and stabilizers in mayonnaise and dressing products. Moreover, Stellan could replace LBG in frozen confectionary products. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. GC-FID Chromatogram of monosaccharide composition of Stellan vs standards Figure 2. Overlay of chromatograms from compositional analysis of alginate and Stellan Figure 3. EI-MS of the 3-O-lact-glu / gal component from compositional analysis. Figure 4. 1H NMR spectra of deacetylated EPS with labelling of the peaks in the anomeric region. B: Enlarged view of 2D HSQC spectra of deacetylated EPS. Figure 5. Spectral overlay of 1H-13C HSQC (purple) and HMBC (green) spectra of Stellan. Figure 6. FT-IR spectra of the EPS extracted from Sphingomonas sanxanigenens Figure 7. Interfacial tension of Stellan vs oil Figure 8. Surface tension of Stellan Figure 9. Shear reversible behaviour of emulsion based Stellan Figure 10. Meltdown performance of the ice cream samples DETAILED DESCRIPTION OF THE INVENTION The present invention describes the chemical structure of a novel exopolysaccharide (EPS); herein so-called Stellan, produced by Sphingomonas sanxanigenens. The inven- tion includes the demonstration of double functionality of Stellan as an emulsifier and texturizer in emulsion systems. Moreover, the use of Stellan in mayonnaise as emulsifier and eggs replacer. The invention also discloses the use of Stellan in frozen desserts as LBG replacer. The invention discloses the functional properties of Stellan. 1) The invention discloses the chemical structure of Stellan Stellan, disclosed herein, is a novel EPS extracted from Sphingomonas sanxanigenens via alcohol precipitation, which has a novel chemical structure composition. Chromato- graphic and spectroscopic analysis reveal an unusual and highly substituted heteropoly- saccharide containing glucose, guluronic acid, and the unusual 3-O-lactyl-hexopyranose in a molar ratio of 2:1:1 as revealed in the structural formula below. Structurally, the main chain of Stellan is composed of a tetrasaccharide as repeating unit. Stellan was found to be highly anionic from uronic acids and an ether linked lactyl group. The molec- ular weight of Stellan is found to be around 500 kDa. The structure is substituted by acetyl and glycerate groups in percent mass 9-18% and 5-12%, respectively. Moreover, Stellan has surface and interfacial tension with values of ≤ 58 mN / m and ≤ 23 mN / m, respectively. Stellan has texturizing properties with gel strength values of at least 120 g. Below the proposed structure of Stellan: Proposed structure for Stellan polysaccharide, composed by glucose, guluronic acid and 3-O-lact-glu / gal in a ratio around 2:1:1. The structure [A-B-C-D] is repeated n times, wherein n is approximately 210 providing a MW of 500kDa. The results disclosed in this patent provide the structural features of a novel EPS; called Stellan, containing a lactyl group in position C3 of one monomeric sugar on the repeating unit. Details of this study are revealed on example 1-2. Stellan could be considered as a new amphiphilic hydrocolloid able to impart double func- tionality, emulsifying by the hydrophobic regions on the structure (lactyl and ester groups) and texturizing by the hydrophilic regions (OH groups). Details on this technical feature are demonstrated in examples 3-4. Stellan demonstrated its mentioned properties in mayonnaise, dressings and frozen des- serts as demonstrated in examples 5-6. The Stellan object of the present invention is produced via aerobic fermentation of Sphin- gomonas sanxanigenens and extracted via alcohol precipitation as per the method de- scribed in example 7. The extracted Stellan powder represent 2 to 3 % of the fermented broth. Stellan is com- posed of carbohydrates in mass percentage of ≥ 70 %, protein ≤ 5 %, minerals ≤ 7 % and polyhydroxybutyrate ≤ 10 %. Values can vary in function to the production process. The Stellan composition is in the range like other commercial polysaccharides such as gellan and xanthan. 2) The invention describes the amphiphilic activity of Stellan Stellan disclosed herein has a surface activity in a concentration higher than 0.05 % showing interfacial tension of 17 mN / m and surface tension of 59 mN / m or less as de- termined according to the method described in example 2. Stellan is considered to be an amphiphilic polysaccharide; that is, a polysaccharide containing both hydrophobic and hydrophilic regions distributed on the polysaccharide backbone. The amphiphilic activity of the polysaccharide is of interest as it can impart interfacial activity such as adsorption at the interface of oil / water droplets and promote emulsification as an example. At the interface of an oil-in-water dispersion, the hydrophilic regions of the polysaccha- ride molecule will be in the water phase while the hydrophobic regions will be in the oil phase. Stellan thereby could form a film around the oil droplets allowing stabilized emul- sions. Thus, Stellan, due to its interfacial activity, can be used as an emulsion stabilizer and texturing agent in food, cosmetics and pharmaceutical products. On the other hand, as described in example 4, the zeta-potential of oil-in-water emulsions prepared with Stellan are stable for at least 1 month. Independently of the oil concentration, a zeta potential value of ≤ -50 mV is measured after 1 month, which is comparable to a con- ventional emulsifier such as Panodan (-69 mV), a diacetyl tartaric acid ester of mono- and diglycerides (DATEM). Other polysaccharides such as high esterified pectin and xan- than shows similar initial z-potential values of ≤ -39 mV and ≤ -51 but oil separation is seen after day 1 resulting in an unstable emulsion. 3) Description of the texturising properties of Stellan Stellan disclosed herein is readily dispersible in aqueous solvents and will hydrate when heated. When hot solutions are cooled, textures varying from viscoelastic fluids to soft, elastic, thermo-reversible gels are formed from 0.03% to 5%. The gels, under certain conditions, can also be shear reversible (Thixotropic). The hydration, gelling and melting temperatures and texturizing properties of the gels are influenced by the Stellan concen- tration, ionic strength, pH, and presence of other ingredients such as sugars, alcohols, fats, oils and proteins. For example, in semi-skimmed milk (1.5% fat) Stellan at 1% will hydrate above approximately 80°C and will form a gel when cooled below 50°C with a gel strength ≥150 g. Said gel will melt when re-heated above approximately 65°C. In standardized tap water Stellan at 1% will hydrate above approximately 70°C and form a gel when cooled below approximately 35°C with a gel strength of ≥100 g. said gel will melt when heated above approximately 50°C. Details are demonstrated in Example 3. 4) The invention discloses the emulsifier properties of Stellan Stellan disclosed herein is a surface-active polysaccharide with interfacial tension of ≤ 23 mN / m able to maintain stable oil-in-water emulsion at dosages from 0.2 - 0.5 % with oil concentration from 5 to 65 %. The oil droplet size distributions vary from 1 to 60 µm depending on oil and Stellan dosage. Emulsions are stable for at least 1 month; maintaining 90 % (Dv (90)) of the total oil volume with particle size below 1µm on day 0 and below 2µm at one month, when 5 % oil was used in the system. Whereas 30 % oil has a broader size distribution, below 21 µm. Moreover, the emulsions present a range of textures from viscous liquids to gels, in function of Stellan usage level (≥ 0.2 %). In addition, thixotropic shear reversible texture is demonstrated for Stellan emulsions preferably between 0.2-0.4 % Stellan with 5-50 % oil. Gels recover at 80 % of the orig- inal strength within 4 hours of being subjected to shear. Details are demonstrated in Example 4. The emulsifier property is of interest due to the enhancement of stability characteristic of hydrophobic compounds such as: soybean, corn, olive, peanut oil namely food oils; castor, squalane, jojoba, and coconut oil namely cosmetic oil; glycerin, diesel, paraffin, N-hexane, xylene namely hydrocarbons. Other polysaccharides such as high esterified pectin or xanthan revealed coalescence and creaming during the first 24 hours which led to phase separation of the oil and water after 1 month. 5) Application of Stellan in emulsion system Mayonnaise and dressing: Stellan disclosed herein can be used to prepare vegan mayonnaise and dressings (vinai- grette or thousand island). Stellan dosage from 0.3 % was able to produce a stable mayonnaise with similar texture to the control sample produced with a multi-ingredient- based emulsifier system. The mayonnaise and vinaigrette are stable for at least one month at pH around 3.8-4.0 with a viscosity of 19000 mPa.s measured with Brookfield, spindle Heipath TB at 10 rpm for 10 seconds. Stellan in this formulation replaces emul- sifier, texturizer and proteins. Details are described in Example 5. Frozen dessert Stellan disclosed herein can function as an alternative to LBG in ice cream production, giving good melting resistance, and a similar ice crystal control benefit as LBG. The Stel- lan gum dosage was 0.10 giving 2.3 % and 0.5 % melting value after 60 min at 20 °C, and ice crystal size distribution 125 and 101.6 µm. Higher dosage > 0.2 % will build texture and the ice cream system will present higher viscosity. In addition to the emul- sifier and texturizing properties of Stellan, this patent describes the ice crystal control effect that Stellan performs in Ice cream. Details are described in Example 6. 7) Description of the production and extraction process of Stellan Stellan disclosed herein is produced via aerobic fermentation of Sphingomonas sanxa- nigenens. The fermented broth has a Brookfield viscosity ≥ 7000 mPa.s. The extraction of Stellan is made by precipitation with hot alcohol to recover approximately 2 to 3 % dry matter. The extraction process follows a common extraction process of EPS by precipitating the broth in alcohol. Details of the process are described in Example 7. EXAMPLES EXAMPLE 1 – Description of the chemical structure and functional groups of Stellan EX 1.1 - Materials: Ethanol (96% and absolute) was purchased from VWR. Methanol, pyridine, acetone, di- methylsulfoxide, and dichloromethane were either anhydrous or analytical grade and ob- tained from Fischer Scientific. Formic acid, acetyl chloride, sodium sulphate, and sodium borodeuteride were obtained from VWR. Sulphuric acid, sodium hydroxide, sodium boro- hydride, polyhydroxybutyrate, deuterated methyl iodide, sorbitol and deuterium oxide were purchased from Merck while acetic acid, acetic acid anhydride, trimethylchlorosilane (TMSCl), and N,O-Bis(trimethylsilyl)trifluoroacetamide (BSTFA) were obtained from Fischer Scientific. All chemicals were used without any purification. EX 1.2 - Methods: 1.2.1 Sugar composition analysis The sugar composition of Stellan was analyzed by GC-FID and GC -TOF-MS following methanolysis and silylation. Briefly, 10 mg of dried stellan was mixed with 1 mL of acidic methanol (0.5 M and 2 M HCl in methanol). The mixture was stirred and heated at 100 °C for 4 h. Pyridine (50 µL) and methanol (1 mL) was added with internal standard (sorbitol, 1 mg / mL) to the cooled mixture; followed by solvent evaporation under nitro- gen at 50 °C. The dried samples were silylated by adding 500 µL pyridine, 250 µL BSTFA, and 50 µL TMSCl. The reaction was carried out for 60 min at 40 °C. The mixture was diluted with 7 mL pyridine and transferred to GC vials. Samples were analyzed by GC- FID (Agilent 7890) and GC-TOF-MS (Agilent 7890, LECO Pegasus® HT) equipped with an Rtx5-MS (Restek, 30m x 0.25mm x 0.25µm). The inlet was operated in split mode (1:20) at 280 °C. The injection volume was 1 µL. Hydrogen was used as carrier gas for GC-FID and helium was used for GC-MS. A ramped flow was used starting at 1 mL / min and increased to 2 mL / min after 10 min. The oven temperature program was started at 115 °C (hold 2 min) and increased 5 °C / min to 240 °C (hold 5 min) giving a total run time of 32 min. The ion source temperature was 230 °C (quadrupole temperature was 150 °C) and the TOF-MS was operated at 20 hz. Monosaccharides were identified from retention time and peak distribution by using standard samples. The monosaccharide standards comprise arabinose, rhamnose, xylose, mannose, glucose, and galactose. 1.2.2. Nuclear magnetic resonance (NMR) Samples were deacetylated by suspending 60 mg Stellan in 50mL of 10mM sodium hy- droxide, the solution was under stirring and heat at 80°C for 2 hours. After deacetylation, Stellan is precipitated in 250 mL ice cold isopropanol, dried overnight at 70°C and grinded prior to NMR analysis. Either 3 mg or 10 mg deacetylated Stellan was mixed with 1 mL D2O at 80°C using a Thermomixer. NMR spectra were acquired using a 600MHz Bruker Avance III NMR Spectrometer equipped with a BBO probe (Bruker BioSpin GmbH, Rhein- setten, Germany) and standard Bruker pulse sequences. NMR analysis were performed at 80°C: 1H NMR (1k scans, relaxation delay 20s), 13C Attached Proton Test (APT; 16k scans), Heteronuclear Single Quantum Coherence (HSQC; 256 scans, acquired size [4k, 512], relaxation delay 2s), 1H-1H Double Quantum Filter - Correlated Spectros- copy (DQF-COSY;256 scans, acquired size [4k, 512], relaxation delay 2s), 1H-1H Total Correlation Spectroscopy (TOCSY; 256 scans; 80 ms mixing time, acquired size [4k, 512], relaxation delay 2s), 1H-1H Nuclear Overhauser Effect Spectroscopy (Noesy; ac- quired size [4k, 512], relaxation delay 2s , 300 ms mixing time), 1H-13C HSQC-TOCSY (256 scans; acquired size [4k, 512], relaxation delay 2s, 80 ms mixing time), Heteronuclear Multiple Bond Correlation (HMBC; 512 scans, acquired size [4k, 512], re- laxation delay 2s, 8Hz or 10Hz long-range coupling constants). Data were analyzed using TopSpin 3.6.5 and MestReNova 14.3.1 software. 1.2.3 Linkage analysis Partially methylated acetylated alditols (PMAA) were prepared with the following method. Approximately 10 mg of Stellan was added to 2 mL DMSO and subjected to sonication for 3 h. Then, 500 µL of sodium hydroxide (10 %) was added to the sample solution and sonicated for 1 h. Deuterated methyl iodide (1 mL) was added to the solution and stirred for 1 h at ambient temperature. The reaction was quenched with 0.5 mL demineralized water and then dialyzed and lyophilized. The reaction was monitored by FT-IR and the cycle was repeated until disappearance of the -OH band (3200-3700 cm-1). Formic acid (1 mL, 90%) was added to the dried methylated sample and stirred for 1 h at 120 °C. The formic acid was evaporated under nitrogen. Trifluoroacetic acid (1 mL, 2M) was added and stirred for 3 h at 120 °C. Excess reagent was removed under nitrogen while also adding toluene. Sodium borohydride / deuteride (500 µL, 0.5 M) was added to the solution and stirred for 3 h at ambient temperature. The reaction was quenched with glacial acetic acid and excess was removed under nitrogen. Acetic acid anhydride (600 µL) and pyridine (150 µL) were added to the solution and stirred for 3 h at 90 °C. Excess reagent was removed under nitrogen. The precipitate was extracted with demineralized water and dichloromethane, which were combined. The organic phase was transferred and washed with demineralized water before being dried with sodium sulphate. The di- chloromethane phase was transferred and removed under nitrogen before being re-dis- solved in 2 mL dichloromethane to obtain the PMAA. The PMAA was analyzed by GC-MS (Agilent 7890, Agilent 5977). Analytes were separated with an Rtx5-MS (30m x 0.25mm x 0.25µm). The inlet was operated in split mode (20:1) at 280 °C and the injection volume was 1 µL. The carrier gas was helium at a constant flow of 1 mL / min. The oven temperature started at 60 °C (hold 1 min), increased 20 °C / min to 130 °C, and then increased 4 °C / min to 290 °C (hold 15 min) giving a total run time of 59.5 min. Detection was carried out in three different ionization modes; 1. EI, 2. PCI (methane), 3. Atmospheric pressure chemical ionization (APCI). For electron ionization the ion source and quadrupole temperatures were 230 °C and 150 °C, respec- tively. For PCI the flow was 15 mL / min and the ion source and quadrupole temperatures were both 150 °C. EX 1.3 - Results 1.3.1. Monosaccharide compositional analyses Methanolysis was used to identify the monosaccharides composition of stellan due to the presence of uronic acids which will be converted to lactones. At a given set of conditions, the ratios between the peaks formed during methanolysis are consistent for the different monosaccharides and in combination with retention times can be used for monosaccha- rides identification. Comparison with monosaccharide standards and Stellan monosac- charides are showed in Figure 1. The analysis showed that Stellan contains glucose (Glu), guluronic acid (GuA), and a hexopyranose with an ether linked lactyl substituent in posi- tion C3. The peak distribution of GuA is typically based on four peaks as the fifth peak cannot be separated from mannuronic acid. The theoretical peak distribution based on alginate did not match the stellan chromatogram due to peak 2 co-eluted with another peak of the unknown component as shown in Figure 2. Adjusting the peak ratio based on the theo- retical value shows a perfect peak distribution for guluronic acid. The quantitative recovery of the monosaccharides was approximately 20%, which may be due to the presence of uronic acids. Generally, the hydrolysis rate is influenced by the monosaccharide composition, anomeric configuration, and glycosidic linkages. The substituted hexopyranose had similar retention time to glucose and glucose ethers indicating a similar mass following derivatization. A high degree of resemblance is seen amongst the EI mass spectra of monosaccharides where the pyranose derivative results in intense fragments of 204 m / z (C2-OTMS + C3-OTMS) and 217 m / z (C1 + C2-OTMS + C3-OTMS). The unknown instead had ions 218 m / z and 231 m / z indicating substitution at C2 or C3 due to the absence of 204 m / z and 217 m / z as highlighted in blue for the structure in Figure 3. The increased fragment mass of 14 indicates that an additional CH2 has been added to a similar fragment with two TMS groups but this can only occur if a CH2group is added on the C2 or C3, which would also make stabilization of the radical unfavorable. Instead, the TMS group is more likely to have been substituted with C4H7O2. The 161 m / z fragment must then either occur from the substituent or be due to a shift in the ionization location. One possible fragmentation would be McLafferty rearrangement if the substituent is placed in the C3 position and ionization occurs on the C2-OTMS group. Additional analysis such as softer ionization mode of PCI indicate that the unknown mol- ecule might correspond to an ether linked molecule and not to an acyl group. Based on the above mentioned, the Stellan structure consists of D-Glucose, L-Guluronic acid and Ether linked sugar. Moreover, the analysis by GC-FID indicates the molar ratio of 2:1:1 / D-Glu:L-GuA:Ether liked sugar). 1.3.2 Nuclear magnetic resonance (NMR) Assignments of deacetylated Stellan were based on 1D 1H and 13C NMR experiment, which are summarized in Table 1. According to the 1D 1H NMR spectra, 5 different 1H NMR signals in the anomeric region were found and labelled as A, B, C, D, and E in Figure 4A. However, as seen from the HSQC spectra only peak A, B, D, and E have a 13C chemical shift in the anomeric region as revealed in Figure 4B. Thus, the repeating unit of Stellan consists of four sugar residues labelled A, B, D, and E, which shows a 1:1:1:1 ratio. Also, a 1:3 ratio was found between each of the sugar residues and the methyl group of the lactyl substituent at 1.36 ppm corresponding to a 1:1 molar ratio (Figure 5A). The methine group of the lactyl substituent was identified at 4.13 ppm. The J-split- ting and the chemical shift indicates residues A and B are in α-conformation, whereas D (1J = 6.80 Hz) and E (1J = 7.09 Hz) are glucose with a J-splitting typical for β-confor- mation. The Guluronic acid was identified as spin system B / C, because of the identifica- tion of a HMBC cross peak between C5 and a 13C chemical shift resonance at 178.0 ppm corresponding to carboxylic acid. Table 1.1H and 13C NMR chemical shift assignment of deacetylated Stellan and important HMBC / NOE correlations Chemical shift for : *correlation not determined due to poor resolution The identified spin systems were connected using NOESY spectra to determine the linkage between the monosaccharides as showed in Table 1. In addition, an HMBC cross peak confirmed ether linked lactyl group substitution at position C3 of sugar residue A as shown in Figure 5. The cross peaks identified that the sugar residues were linked as follows: A1-E3, B1-D4, D1-A4, and E1-B4, which are in agreement with the sequence: →4) β-D (1→4) α-A (1→3) β-E (1→4) α-B / C (1→. The repeating unit could therefore be identified as: →4) β-Glu (1→4) α-3-O-Lac-Glu / Gal (1→3) β-O-Ac-Glu (1→4) α-GuA (1→, which is in agreement with the analysis for monomeric composition, PMAA, and LC-MS / MS. 1.3.3 Linkage determination The results showed that the two most dominant peaks correspond to 1,3-Glu and 1,4- Glu, which were equally abundant. Significantly less of 1,2-Glu was identified and was not included in further structures (1:10 to 1,3-Glu). EXAMPLE 2 Fourier Transform Infrared Spectroscopy (FT-IR) of Stellan EX 2.1- Material: Ethanol, stellan, sanxan, gellan powder. EX 2.2 -Method 2.2.1 Fourier Transform Infrared Spectroscopy (FT-IR) A Bruker Alpha platinum attenuated total reflectance Fourier Transform Infrared (ATR- FT-IR) spectrophotometer was used to record 64 scan spectra from 4000 to 400 cm-1 with resolution of 2 cm-1. The ATR crystal was cleaned with ethanol and solid powder of Stellan samples were compressed against the diamond crystal. EX 2.3 – Results 2.3.1. FT-IR spectroscopy analysis The infrared spectra in the range 4000 to 400 cm-1 are given in Figure 6; showing au- thentic functional groups of Stellan. Thus, a broad stretching vibration of the O-H at 3281 cm-1, and the C-H vibration of the aliphatic methylene at 2920 were revealed. A strong absorption at 1723 cm-1 corresponding to the stretching vibration of carboxylic ester (C=O) revealed the presence of polyhydroxybutyrate (PHB). The band 1600-1610 were suggested as the O-C-O carboxylate asymmetric stretching. The bands located at 1379 and at 1249 cm-1 were assigned to the C-OH stretching vibration with implication of the O-C-O symmetric stretching vibration. The peak at 1042 is attributed to the C-O- group of polysaccharides. The peak at 980 could correspond to the C-O vibration of the gulu- ronic acid. Interesting our results varies from the previous publication by Huang et al.11 where the authors reported the spectra of sanxan EPS produced by Sphingomonas sanxanigenens with main peaks on 1637 cm-1, 1413 cm-1, 1081-1037 cm-1. It is a possibility that the stretching vibration of the functional groups could have shifted, but it is evident that Stellan shows original peaks in 1379 and 1249 cm-1 as an indication of the novelty of the polymer. It is worth nothing that peaks in 1637 and 1249 cm-1 are not present in Sanxan, Gellan or Xanthan. EXAMPLE 2a- Amphiphilic properties of Stellan The interfacial and surface activity of Stellan was determined by the plate Krüs standard plate- K100 force tensiometer. The data in Table 2 and Figure 7-8 shows that the Stellan has surface and interfacial activity, as demonstrated by the reduction of surface and interfacial tension. Gellan and Xanthan were included as reference samples. Surface Ten- Interfacial Sample sion at 25ﹾC Tension at 25 (mN / m) ﹾC (mN / m) Stellan 53 17 Xanthan 48 23 Gellan 67 27 Table 2- Surface and Interfacial tension values for Stellan solution with 0.05 % concen- tration. EXAMPLE 3 - Textural properties of Stellan EX.3.1 Materials Milk (1.5% fat), deionised water, standardised tap water (STP) composed by 1,4g NaCl, 0,3g CaCl22H2O , 998,3g deionized water. Sucrose and soya protein (Supro TX221D). EX. 3.2. Methods 3.2.1 Preparation procedure in Milk and STP 5 g of deionised water (to account for evaporation) was added to 742.5g of milk or STP in a beaker. The appropriate weight of Stellan powder was then added at room temper- ature whilst mixing at 600 rpm with a 65 mm deflocculating blade propeller. After ap- proximately 30 seconds the speed was reduced to 400 rpm and mixed for a further 2 minutes. The dispersion was then placed in a water bath at 94 – 95 °C and stirred at 400 rpm for 15 minutes. Stirring was stopped after 15 minutes and any foam was removed before pouring the solution into 3 glass dishes of 80 mm diameter and 45 mm height. After 10 minutes the dishes were covered with a lid. The dishes were then stored for 22 to 25 hours at room temperature before the texture was measured. 3.2.2. Preparation procedure in 60 % sucrose 480 g of sucrose, 316 g of STP and 4.5 g of deionised water (to account for evaporation) were placed in a beaker and mixed at 600 rpm with a 65 mm deflocculating blade pro- peller for approximately 2 – 3 minutes to dissolve the sucrose. The appropriate weight of Stellan powder was then added to room temperature whilst mixing at 600 rpm with a 65 mm deflocculating blade propeller. After approximately 30 seconds the speed was re- duced to 400 rpm and mixed for a further 2 minutes. The dispersion was then placed in a water bath at 94 – 95 °C and stirred at 400 rpm for 15 minutes. Stirring was stopped after 15 minutes and any foam was removed before pouring the solution into 3 glass dishes of 80 mm diameter and 45mm height. After 10 minutes the dishes were covered with a lid. The dishes were then stored for 22 to 25 hours at room temperature before the texture was measured. 3.2.3. Preparation procedure in 3 % soya protein 579 g of STP and 4 g of deionised water (to account for evaporation) were added to a beaker and placed in a water bath at 80 °C. 18 g of soya was then added whilst mixing at 700 rpm with a 65 mm deflocculating blade propeller for 2 minutes. After 2 minutes the speed was reduced to 400 rpm and mixing was continued for a further 13 minutes. The speed was then increased to 700 rpm and the appropriate weight of Stellan powder was added. After approximately 2 minutes the speed was reduced to 400 rpm and mixed for a further 13 minutes. Stirring was then stopped and any foam was removed before pouring the solution into 3 glass dishes of 80 mm diameter and 45 mm height. After 10 minutes the dishes were covered with a lid. The dishes were then stored for 22 to 25 hours at room temperature before the texture was measured. 3.2.4. Measurement of gel texture. Gel texture was measured using a TAXT2 texturometer (Stable Micro Systems) fitted with a 0.5 inch cylindrical probe programmed to penetrate the gel at 1 mm / s to a depth of 30 mm. The trigger force was set to 1 g. The maximum force (g) was recorded as a measure of the gel strength of the gels. The distance (mm) at maximum force was recorded as a measure of the gels elasticity / brittleness and the force (g) at 10 mm was recorded as a measure of the firmness of the gel. Results are recorded as the average of the 3 gels tested. 3.2.5. Measurement of gelling and melting temperatures Gelling and melting temperatures were measured using an Anton Paar MCR 501 in oscil- latory shear mode fitted with a cup and bob geometry with an effective bob length of 40 mm and diameter of 26.66 mm and a cup diameter of 28.93 mm. The cup was heated to 95°C before being filled with the hot solution (90°C). The Bob was lowered, and the liquid surface was covered with a layer of silicone oil to prevent evaporation before the lid was placed over the cup. The sample was then cooled from 95 to 20 °C at 3°C / min whilst measuring the storage (G’) and loss (G”) moduli at a frequency of 1 Hz and a strain of 1%. Gelling was defined as the temperature at which G’ becomes greater than G”. The sample was the heated from 20 to 95°C and melting was defined as the temperature at which G” becomes greater than G’. The soya protein sample was cooled and heated between 80 -20°C. EX.3.3 Results The results demonstrate that Stellan, disclosed herein, is able to form free standing gels in a variety of media at concentrations from 0.5%. The gel texture, gelling temperature and melting temperature results are demonstrated in Table 3. The texture can be de- scribed as soft and elastic, and gels are thermoreversible. Stellan Medium Maximum Distance at Force at Gelling Melting (%) force maximum 10 mm. temper- temper- (g) force (g) Firm- ature ature Gel (mm) Elas- ness (°C) (°C) strength ticity / Brit- tleness 0.5 Milk 58..4 23.9 12.9 49.8 66.3 1.0 Milk 168.8 25.1 38.5 52.5 70.1 0.5 STP 53.6 25.1 11.4 43.0 59.7 1.0 STP 128.7 26.1 25.6 47.7 63.5 0.5 60% sucrose 123.6 30.0 16.8 56.9 74.5 1.0 60% sucrose 216.8 30.0 37.0 61.5 77.7 0.5 3% soya 39.5 22.4 14.9 41.4 59.3 1.0 3% Soya 93.1 20.9 35.6 47.6 63.9 STP= Standardized tap water Table 3- Texture, gelling and melting of Stellan gels in different media. EXAMPLE 4 - Emulsifying properties of Stellan EX 4.1 - Materials Rapeseed oil, water, gellan, xanthan, pectin, and Panodan (diacetyl tartaric acid esters of mono- and diglycerides). EX 4.2 – Methods 4.2.1 Emulsion preparation Emulsions O / W were prepared in two steps: Step 1- stock sample: 1 g of dried Stellan was dispersed in 100 mL of deionized water and maintained under stirring for 15 minutes at room temperature. The dispersed Stellan was then transferred to the water bath (95 °C) for 15 min. The obtained solution was cooled down to room temperature and stored overnight. The resulting stock Stellan gel was used in the subsequent tests. Step 2- Emulsion preparation: Emulsions were prepared by varying oil, water and the stock Stellan gel concentration as indicated in Table 4. The stock Stellan gel and the required amount of water are mixed using an Ultra-Turrax (IKA T25) at maximum capacity for 5 min, after this time the oil is incorporated and mixed for an additional 5 minutes. The resulting emulsions were ho- mogenized at room temperature by using a homogenizer (GEA Niro Soavi Panda PLUS) at 300 bar. Samples were taken after the 3rd recirculation. Samples were recovered in test tubes and stored in a refrigerator and evaluated over a 1 month period. Other samples such as pectin and xanthan were also previously hydrated for example xanthan was hydrated at room temperature for 30 minutes and Pectin were dispersed in hot water (85 C) stirred for 30 minutes. Samples Stabilizer O / W Emulsion characteristic day 0 (%) Oil content (%) Stellan SE6 0.3 5 White fluid 0.3 30 Soft white gel with thixotropy behavior 0.4 30 Soft white gel with thixotropy behavior 0.5 30 Soft white gel Pectin RS 0.3 5 White fluid 30 Unstable due to flocculation and coales- cence Xanthan 0.3 5 White fluid 30 Unstable due to flocculation and coales- cence Panodan 0.3 5 White fluid 30 Unstable due to flocculation and coales- cence O / W = Oil in Water emulsion Table 4- Emulsion composition of test samples The Stellan samples were compared to samples prepared with other polysaccharides and an emulsifier (Panodan) for their emulsion quality, stability, and texture analysis. 4.2.2 Determination of droplet size and distribution by Malvern Master- sizer The droplets size and distribution of the O / W emulsion were measured by a Malvern Mastersizer 3000 laser diffraction equipment with a Hydro cell, according to method 23.8220.22, rev. 9 (IFF). Briefly, 1 drop of emulsion was dispersed in 20 mL of MilliQ water and stirred at room temperature for two hours. Analysis was performed when 5 to 8 % obscuration is reached by dispersing the emulsion samples in the mastersizer solu- tion (MilliQ water pH 4); while using an He-Ne laser / red light source at 632.8 nm and blue light source at 470 nm for wet measurements (suitable for the smallest particles). Analysis is performed in 30 min. 4.2.3 Z-potential The zeta potential was measured on Zetasizer Nano-ZS according to method 23.8220.01, rev. 7 (IFF). Briefly, one drop of emulsion was diluted in 5 mL of MilliQ water which contains one drop of eye solution (0,9 % of sodium chloride, phosphate dihydrate / po- tassium dihydrogen phosphate) to make pH 7 solution. Measurements were recorded using He-Ne laser at 633 nm. The particles move in an electric field with a velocity related to their zeta potential. From this, the zeta potential can be calculated (Stern model / shear plane). Note that dilution of emulsion samples is dependent on the application and typi- cally varies between 1:100x and 1:200x. 4.2.4 Rheology Viscosity was measured using a HAAKE MARS II rheometer with a UTC temperature con- troller and a MARS II control unit. Measurements were made on two parallel plates con- sisting of a 60-mm stainless steel stationary plate, and a 35mm stainless steel plate (PP35Ti) as rotor. The temperature for measurements was 20°C and the gap size be- tween plates set at 1.0mm. Thixotropy was measured with a, Anton Paar MCR 502 rheometer fitted with a concentric cylinder (CC27) after 38 days storage at 4°C using repeated cycles of shear / gel recovery as follows. The emulsion was loaded onto the rheometer at 20°C and subjected to a shear rate of 500 s-1 for 10 minutes. The storage modulus (G’) was then measured for 4 hours at 1 % strain and a frequency of 1 Hz. A data point was taken every 0.8 minutes. After 4 hours the sample was sheared again at 100 s-1 for 10 minutes followed by measurement of the storage modulus for 4 hours. This shear and storage modulus measurement was then repeated. In total the sample was subjected to 3 shear / gel recovery cycles. Tan delta was calculated by dividing the G” value by the G’ value. Recovery (of the gel strength after shear) was calculated by comparing the G’ values at the start and end of shear cycles 2 and 3 with the value after 4 hours in Cycle 1 and are expressed as a percentage. EX 4.3 – Results The particle size distribution of the emulsion droplets was controlled by Malvern Master- sizer instrument. Thus, 90 % (Dv (90)) of the oil droplets has a size of 2 µm or smaller, when 0,3 % Stellan and 5 % oil were used. Higher dosages of Stellan and oil reveal bigger oil droplets with values below 40 µm. Results are summarized in Table 5. Pectin, on the other hand display small oil droplets below 3 µm but those systems are not stable over time reaching coalescence and flocculation as in the case of xanthan. Importantly, the stability of the oil droplets for 5% oil emulsion was confirmed as well as the variation in mean droplet size in the 30 % oil emulsion. It is remarkable that only Stellan imparts emulsifying stabilizing and thickening properties; as is very well-known emulsion are generally stabilized by surfactants, amphiphilic polymers, lipids and proteins due to the formation of the interfacial film. Therefore, it is reasonable to think that Stellan could impart certain functionality at the hydrophobic and hydrophilic layer. Samples Dos- O / W Oil droplets mean diameter (µm) Visual appear- age Oil ance (%) con- tent Day 0 1 Week 1 Month 1 month (%) Dv(90) (µm) Dv(90) Dv(90) (µm) (µm) Stellan SE6 0.3 5 1 1,7 2 Stable emulsion 0.3 30 7 14 21 Stable emulsion 0.4 30 14 38 40 Stable emulsion 0.5 30 15 x x Stable emulsion Pectin RS 0.3 5 2 3 nd Oil separation 0.3 30 39 nd nd Oil separation Pectin YF 0.3 30 175 nd nd Oil separation Xanthan 0.3 5 5 nd nd Oil separation 0.3 30 132 nd nd Oil separation Panodan 0.2 5 2 2 2 Creaming 0.2 30 149 nd nd Creaming X= data not recorded due to contamination, however qualitative analysis indicate stable emulsion. Nd= analysis were not recorded due to creaming or oil separation Table 5- Size distribution of emulsion droplets meassured at day 0, week 1 and month 1. The surface charge of the oil droplets was measured by ζ potential. The result shows that Stellan emulsions present a stable charge (-56 mV) over one month with no visible creaming as indicated in Table 6. Pectin and Xanthan, on the other hand, show a similar initial charge to Stellan, however the resulting emulsions were unstable and zeta poten- tial was not measured due to phase separation. Panodan an emulsifier based on diacetyl tartaric acid ester of mono-diglycerides (DATEM) showed stable values but higher than Stellan (-70 mV) indicating a higher surface tension but showed coalescence after one week for a 5 % oil emulsion and creaming at higher 30% oil within the first day. The results demonstrate that Stellan can both form emulsions and stabilize said emul- sions. Samples Dosage O / W Zeta potential (mV) Visual appearance (%) Oil con- tent (%) Day 0 1 1 1 month Week Month Stellan SE6 0.3 5 -51 -50 -51 Stable 0.3 30 -52 -51 -51 Stable 0.4 30 -54 -56 -56 Stable 0.5 30 -55 -56 -56 Stable Pectin RS 400 0.3 5 -37 -37 nd Oil separation 0.3 30 -39 nd nd Oil separation Pectin YF 575 0.3 30 -51 nd nd Oil separation Xanthan 0.3 5 -51 nd nd Oil separation 0.3 30 -47 nd nd Oil separation Panodan 0.2 5 -69 -70 -69 Creaming 0.2 30 nd nd nd Creaming O / W= Oil-in-Water emulsion Nd= not determined due to creaming on the samples Table 6- Surface charge values determined by ζ-potential. Rheological characterization results of the emulsions are given in Table 7. They demon- strate that the emulsions have a gel-like texture with thixotropic behaviour. The gel strength of the emulsions, (as measured by Storage modulus (G’), was found to increase in the first 4 hours after removal of the shear to give a gel-like texture with (G’) being at least 10 times higher than the Loss modulus (G”) as demonstrated by the Tan delta (G” / G’) being less than 0.1. It is generally recognised that tan delta values of less than 1 indicate gel-like characteristics. As demonstrated in Figure 9. Re-application of shear at a rate of 500 s-1 for 10 minutes reduced the gel strength of the emulsions to less than 27% of their original value however, at least 80% was recovered within 4 hours of re- moval of the shear in cycle 2 and cycle 3 with a tan delta after 4 hours below 0.1. The ability of the gel strength to recover and tan delta to be below 0.1 after repeated shear cycles demonstrates the thixotropic (shear reversible) property of the gelled emulsions prepared with Stellan. Emulsion Cycle 1 Cycle 2 Cycle 3 sample Data 4 0 4 hours hours hours 0 hours 4 hours G" (Pa)1.37 2.40 1.42 2.46 1.49 0.2%, Stellan, G' (Pa) 24.29 5.96 22.06 6.13 20.64 6% oiltan delta0.06 0.40 0.06 0.40 0.07 Recovery (%) - 24.5 90.8 25.3 85.0 G" (Pa)0.82 1.71 0.91 1.86 0.98 0.3% Stellan SE6,G' (Pa)11.37 2.62 11.30 3.01 11.99 5% oïl tan delta 0.07 0.65 0.08 0.62 0.08 Recovery (%) - 23.0 99.3 26.4 105.5 G" (Pa) 2.89 4.56 2.81 4.57 2.76 0.2% Stellan SE6, G' (Pa) 34.64 6.56 31.87 6.93 29.67 30% oïl tan delta 0.08 0.70 0.09 0.66 0.09 Recovery (%)- 17.9 87.0 18.9 81.0G" (Pa)1.75 3.60 1.96 3.66 2.03 0.2% Stellan S6, G' (Pa) 24.43 5.95 23.11 6.08 21.63 30% oïltan delta0.07 0.61 0.08 0.60 0.09 Recovery (%)- 24.4 94.8 24.9 88.5G" (Pa)4.06 8.15 4.08 8.58 4.27 0.3% Stellan S6, G' (Pa) 80.57 17.99 76.88 20.60 78.69 30% oïltan delta0.05 0.45 0.05 0.42 0.05 Recovery (%) - 23.3 95.4 25.6 97.7 Table 7-Rheology characterisation results of Stellan emulsions. Conclusion This example demonstrates that Stellan disclosed herein at dosages from 0.2 - 0.5 % can be used as oil stabilizer or emulsifier in oil-in-water (o / w) system; thus, Stellan could replace chemical surfactants and other modified polymers in emulsion systems. Moreover, the emulsions present thixotropic rheological behaviour whit a range textures from viscous liquids to gels, depending on Stellan usage level (≥ 0.2%) EXAMPLES 5 – Stellan in Mayonnaise EX 5.1 – Material and methods Mayonnaise having an oil concentration of 50 % (w / w) was prepared following on the basis of the formulation described in Table 8. Trial 1 Trial Trial 4 Trial 5 (g) 2(g) Trial 3(g) (g) (g) Ingredient 0.3% re- 0,3 0,4 %- gel p % dr 0,3 %- in Oil gel pre- Powder pared y mix pared Tap Water775,8 775,8 775,8 774 474,1Rapeseed oil900,0 900,0 900 900 550Sugar36,0 36,0 36 36 22Vacuum Salt18,0 18,0 18 18 11Potassium sorbate, Granulate 1,8 1,8 1,8 1,8 1,1 Mustard, french Classic Yellow 27,0 27,0 27 27 16,5 Stellan SE65,4 5,4 5,4 7,2 3,3Vinegar organic 12% 36,0 36,0 36 36 22 Total Batch Size1800,0 1800,0 1800,0 1800,0 1100,0Table 8- Mayonnaise formulation using Stellan as emulsifier and stabiliser The procedure used to produce mayonnaise was as follows: - For trials 1 and 4 - Stellan was dispersed at room temperature in a portion of the water for 15 minutes and then heated at 95°C for 15 minutes. Then the solution was cooled down to room temperature (23°C) at which gel structure starts to develop. - Mix the Stellan gel with the remainder water in a thermomixer and blend for 5 minutes. - Add sugar, salt and potassium sorbate to the thermomixer and mixed for 4 minutes at speed 8. - Slowly added the oil and continue mixing for 3 minutes at speed 7 - Vinegar and mustard were added and mixed for 15 sec. at speed 4.5 - Trials 2 – The Stellan was dry mixed with the sugar, salt and potassium sorb- ate. - The dry blend was added to the boiling water and mixed until all ingredients were dissolved, (approximatively 1 minute at speed 8 in the thermomixer). - The solution was cooled down to approximatively 20°C and mixed for 30 sec. at speed 8. - The oil was added slowly during 3 minutes at speed 7 - The vinegar and mustard were added and mixed for 15 sec. at speed 4.5 - Trials 3 – Stellan was dispersed with oil. Despite the good dispersion, stellan become insoluble when water was added to the system, showing swelled par- ticles. This process showed heterogeneous system and no homogeneous emul- sions. - Trials 5 – Stellan was dispersed in cold water with all dry ingredients. This process resulted on a phase separation during the first hour as an indication of the inactivity of Stellan as emulsifier or stabilizer when is dispersed in cold water. EX 5.2 – Results The pH and Brookfield viscosity, (Spindle Heipath TB at 10 rpm for 10 seconds), of the mayonnaise product were controlled over one month as shown on Table 9. The pH after one month storage at 5°C remains the same as measured on day 0 (around pH 3.9). The viscosity after storage at refrigeration (4 °C) for 1 month was 11000 mPa.s and 19000 mPa.s for 0.3 % and 0.4 % Stellan, respectively. The qualitative analysis after one month demonstrates a stable and homogeneous mayonnaise. It is remarkable that Stellan can behave as an emulsifier and a texturant, interesting to replace commercial emulsifiers. Mayonnaise pH Brookfield viscosity (mPa.s) Day 0 Week 1 Month 1 Day 0 Week 1 Month 1 Trial 1 3,95 3,85 4,02 11220 13000 11880 Trial 2 3,96 3,86 3,95 10680 12800 13520 Trial 3 - - - Trial 4 4,01 3,8 3,93 19340 20160 215629 Trial 5 - - - Reference* 3,9 118000 * reference mayonnaise were produced using an emulsifier system containing (68 % Guar gum, 18 % Xanthan, 17 % protein). No values for Trial 3 and 5 as no stable emulsion was obtained. Table 9 – pH and Brookfield viscosity of Mayonnaise produced with Stellan as emulsifier EXAMPLES 6 – Stellan in frozen dessert EX 6.1 – Material and Methods Ice cream having a vegetable fat concentration of 8 % (w / w) was prepared following on the basis of the formulation described in Table 10. Reference ice cream sample no. 1 was made with 0.20 % locust bean gum (GRINDSTED® LBG 246), and the Stellan was tested in 3 dosages: 0.06 % (ice cream sample no. 2), 0.10 % (ice cream sample no. 3), and 0.30 % (ice cream sample no. 4). Ingredient / Tri- als 1 (%) 2 (%) 3 (%) 4 (%) Water (Tap) 63.49 63.63 63.59 63.39 Refined coconut oil 8 8 8 8 Skimmed milk powder 9.3 9.3 9.3 9.3 Whey powder 2.5 2.5 2.5 2.5 Sucrose 12 12 12 12 Glucose syrup powder 32 DE, 4 4 4 4 95% TS. CREMODAN® SUPER SP VEG 0.3 0.3 0.3 0.3 NP GRINDSTED® LBG 246 0.2 0 0 0 Vanilla Flavor SC905821 0.2 0.2 0.2 0.2 Bright'nYELLOW Annatto 2.5 LWS- 0.01 0.01 0.01 0.01 AS Stellan SE6v4 Eln:20220223- 0 0.06 0.1 0.3 016 Trial total % 100 100 100 100 NP= Emulsifier, mono-and diglycerides of fatty acids Table 10- Ice cream formulation using Stellan as LBG replacer. The production process follows as indicated below: - Melt the fat at 50 °C - Mix liquid ingredients at 20-22 °C - Mix dry ingredients and add to the water phase at 20-22 °C - Add flavoring and coloring - Add the fat and increase temperature to 70 °C - Homogenize the mixture at 78 °C / 175 bar - Pasteurize at 84 C / 30 sec - Ageing at 5 C for minimum 4 hours - Freezing and light extrusion with 100 % overrun - Fill - Freezing in hardening tunnel at -30 °C - Storage at -25 °C Ice cream were analysed by rheology, meltdown determination, Heat shock treatment, and Ice crystal size distribution. Viscosity Mix viscosity was determined by Brookfield method using: Disc L2, time: 30 sec., speed 30 rpm, temperature: 5°C), results are in mPa.s. Viscosity was measured the day after mix-production. Flow-curve Mix flow-curve measurement was done according to IFF Brabrand Departmental Instruc- tion No. 23.8510.121 from IFF Nourish and done by means of MCR, Modular Compact Rheometer. Meltdown Determination The melting rate (drip rate) was done according to Technical Memorandum No. 2520 from IFF. Briefly, A rectangular piece of ice cream (125 cc, dimension: approx. 100 mm x 50mm x 25 mm), which has been tempered to approx. –18°C for a minimum of 24 hours, is weighed and placed on wire netting. The room, in which the melting process takes place, is kept at a constant temperature of 20 °C + / –1 °C. The wire netting is placed above a 500 ml glass beaker placed on an analytical balance. The analytical bal- ances are connected to a computer which registers the weight of the ice cream every 2 minutes and calculates the percentage of melted ice cream as a function of time. After 120 minutes a graph of the melting behaviour can be plotted as revealed in Figure 10. The time elapsed before the first drop of liquid is released from the product is also rec- orded. Heat shock treatment The heat shock treatment was done according to Technical Memorandum no. 2524 from IFF. Briefly, the products were tempered and stored in a freezer cabinet at -18°C. The tempered products were placed in a heat shock freezer cabinet with a temperature var- ying between -20°C and -5°C every 6 hours. The products were kept in this freezer cabinet for 7 days. All samples, both fresh and heat shock-treated, were tempered at - 18°C for 2 days before being analysed qualitatively for iciness (coarse and gritty), sand- iness (lactose crystals that appear to be insoluble in the mouth), shrinkage (reduction in product volume due to air loss) and serum separation. Determination of Ice Crystal Size Distribution Ice crystal size distribution was determined according to Technical Memorandum No. 2522 from IFF. Briefly, on a steel board inside the glove box, a pinch of ice cream (heat shock treated) was suspended in one drop of n-butanol and dispersed by squeezing be- tween two microscope slides until the sample appears homogeneous to the eye. This sample preparation results in a monolayer of ice crystals with a few per cent of overlap- ping crystals. The air bubbles are largely removed by the n-butanol treatment. Around 150 to 300 crystals are measured for each crystal size distribution analysis. EX 6.2 – Results Stellan can function as an alternative to locust bean gum in ice cream application (high melting resistance and good heat shock protection). The dosage of Stellan will be below 0,3 % as higher concentrations provide high viscosity mix going to gelling which are not optimal for the ice cream process. The meltdown performance revealed that ice cream prepared with Stellan had almost no drip after 2 hours. This high melting resistance is similar to that seen with LBG, a tradi- tional ice cream stabiliser. The Ice crystal size distribution analysis showed that Stellan, at a dosage between 0.1 to 0.3%, has a similar ice crystal control effect as 0.2% LBG. Optimal Stellan dosage is to be found between 0.10 and 0.30%. Ice cream mix viscosity: Ice cream mix viscosities are shown in Table 11 (Brookfield). Stellan gave higher mix viscosity than 0.20% locust bean gum, except when dosed at 0.06%. The higher the Stellan dosage, the higher the mix viscosity. At 0.30% Stellan, the ice cream mix was gelled. Sample / Trial Hydrocolloid - % (w / w) Viscosity in Ice cream mix (mPa.s) 1 Grindsted LBG 246 – 0.2 181 2 Stellan – 0.06 240 3 Stellan – 0.1 622 4 Stellan – 0.3 Gel* *samples were to gelled for measuring Table 11 – Brookfield viscosity of ice cream mix Meltdown performance Stellan gave similar high melting resistance as LBG as demonstrated in Table 12. At 0.30% Stellan dosage, the ice cream had almost no drip even after 2 hours as demons- trated in Figure 10. Sample / Trial Hydrocolloid First drop Melted after 60 min (% w / w) (min) (%) 1 Grindsted® LBG 246 – 21 4,1 0.2 2 Stellan – 0.06 21 2,3 3 Stellan – 0.1 44 0,8 4 Stellan – 0.3 55 0,5 Table 12. First drop and melted ice cream after 1 hour Ice crystal size analysis Ice crystal size distribution in the 7 day heat shock treated ice cream samples is shownin Table 13. TheIce crystal size distribution analysis demonstrates that Stellan can per- form a similar ice crystal control effect as 0.2% LBG. Optimal Stellan dosage is to be found somewhere between 0.10 and 0.30%. Sample / Trial Volume fraction Volume fraction Volume fraction D(10.3) D(50.3) D(90.3) 1. 0.2% LBG 64.8 µm 94.5 µm 118.5 µm 2. 0.06% Stellan 64.5 µm 99.3 µm 125.6 µm 3. 0.1% Stellan 65.6 µm 98.9 µm 131.0 µm 4. 0.3% Stellan 55.1 µm 79.7 µm 101.6 µm Table 13. Ice crystal size distribution in ice cream samples after the 7 day heat shock test EXAMPLES 7 – Stellan fermentation and extraction EX 7.1 – Material and Methods Sphingomonas sanxanigenens, culture medium composed of saccharose, yeast extract, malt extract, soy peptone; culture medium containing glucose, yeast extract, magnesium sulfate, dipotassium phosphate, sodium nitrate, sodium chloride and sulfate ferric. Fermentation process was followed as described in prior art. Briefly, S. sanxanigenens was cultured exponentially in a nutrient medium of: saccharose 40 g / L, yeast extract 3 g / L, malt extract 3 g / L, soy peptone 5 g / L. this was then inoculated into the fermentation medium containing: 41 g / L of glucose, 0,20 g / L yeast extract, 1,05 g / L magnesium sulfate (MgSO4.7H2O), 1,2g / L , 1,20 g / L dipotassium phosphate; 2,60 g / L of sodium nitrate (NaNO3); 0,40 g / L of sodium chloride; 0,005 g / L sulfate ferric (FeSO4), 1,00 g / L CaCO3. The pH of the medium was adjusted and maintained to 7.5. The fermentation was under aerobic conditions, with agitation at 220 rpm, at 30° C for 72 hours. Stellan extraction: The extraction process follows various steps going from a dilution of the broth, killing bacteria, precipitation, washing, drying and milling process. Briefly, 150 g of broth was diluted with 30 g of deionized water, heated at 100° C for 30 minutes. The hot mixture is then added to 400 mL Isopropanol (IPA) which was previously heated at 80° C. The Stellan fibers are pressed and washed twice on 70 % isopropanol. The washed fibers are finally dried at 40°C for 15 hours. EX 7.2 – Results The yield of Stellan powder recovered after milling process reaches 2 to 3 %, depending on the fermentation broth, where viscosity is measured using a Brookfield viscometer (spindle S64 at 30 rpm for 30 sec). The fermented broth usually have viscosities ≥7000 mPa.s. The invention provides a compound obtainable by the process comprising the steps of (A)fermenting S. sanxanigenens under aerobic conditions at 30° C for 72 hours, (B) heating the broth to 100° C for 30 minutes, (C) extracting with isopropanol at 80° C, and (D) optionally drying and milling.

Claims

CLAIMS 1. A chemical compound of a novel polysaccharide characterized by the structural formula below:wherein R could be a hydroxyl, acetate or glycerate group; in which the compound contains a tetra polysaccharide as a repeating unit holding a lactyl group in posi- tion C3 of a monomeric sugar.

2. The compound, according to claim 1, wherein the glucose, guluronic acid and 3- O-lact-glu / gal are present in a ratio of around 2:1:

1.

3. The compound, according to any of the claims 1-2, wherein molar mass is at least 500 kDa.

4. The compound, according to any of the claims 1-3, wherein the acetyl and glyceryl groups are present in a percent mass between 9-18% and 5-12 %, respectively.

5. The compound according to any of the claims 1-4, present original FT-IR spectra peaks at 1379 and 1249 cm-1 region.

6. The compound, according to any of the claims 1-5, wherein the compound is an amphiphilic hydrocolloid able to impart double functionality of emulsifying and texturizing.

7. The compound, according to any of the claims 1-6, wherein the compound has a surface tension value up to 58 mN / m.

8. The compound, according to any of the claims 1-7, wherein the compound has an interfacial tension value up to 23 mN / m.

9. The compound, according to any of the claims 1-8, wherein the compound at 1% in 1.5% fat milk has a gel strength of at least 120 g.10.An emulsion composition with texturizing and emulsifying properties containing at least 0.2% of the compound according to any of the claims 1 to 9, between 1 to 50 % of oil in water. 11.The emulsion composition, according to claim 10, wherein the oil is a food oil preferably rapeseed, sunflower, corn in a preferred amount between 5 to 50 %. 12.The emulsion composition, according to any of the claims 10-11, wherein at least 90% of the oil droplets are below 2µm after a period of at least 1 month. 13.The emulsion composition, according to any of the claims 10- 12, wherein the texture is shear reversible with recovery of at least 87 % using a preferred amount between 0.2-0.3 % of the compound as described in claims 1 to 9.

14. The emulsion composition, according to any of the claims 10 to 13, wherein the emulsion is stable after 1 month with a Zeta potential below -50mV. 15.Use of the compound as defined in any of the claims 1 to 9 in food applications. 16.The use according to claim 15, in frozen desserts, mayonnaise or dressing. 17.The use according to claim 16 in frozen desserts, wherein the compound is present in an amount between 0.10 and 0.30 %, giving resistant to melting with values between and 0,5 % and 2,3 % and ice crystal size distribution between 101,6 and 125 µm. 18.The use according to claims 15-16, in mayonnaise and dressing, wherein the com- pound is present in an amount above 0.30 %, and providing a viscosity at about 19000 cP, and a pH between 3.8 and 4.0.

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