Gelled connective tissue analogue for use in plant-based products

Abstract

The invention relates to a method of making a gelled connective tissue analogue for a food product, said method comprising preparing a dispersion of a konjac glucomannan source, a carrageenan source, and a monovalent cation salt in water; optionally adding lipid as oil and / or melted fat to the dispersion, for example sunflower oil, and emulsifying to create an emulsion; hydrating the konjac glucomannan source and carrageenan source by agitating, for example for at least 5 minutes; heating to functionalize the konjac glucomannan source and carrageenan source; and cooling to form a gelled connective tissue analogue.

Description

[0001]Gelled connective tissue analogue for use in plant-based products Introduction Connective tissue analogues are used in plant-based meats to bind together several layers of plant protein extrudate slabs. This creates a cohesive plant-based meat analogue while preserving firmness, chewiness and fibrousness which are essential features for a “meat-like” texture. Egg white is often used as a connective tissue analogue. However, egg white is not vegan and it does not bring any added value in terms of juiciness and fatty mouthfeel which are important textural markers of high grade meat quality. There is a strong need to find new connective tissue analogues which enhance these key sensorial properties for next generation meat analogues. Summary of the invention The present invention uses cold-set carbohydrate gel technology as a connective tissue analogue for plant-based meat analogues. The invention relates to a method of making a connective tissue analogue, said method comprising preparing a dispersion of a konjac glucomannan source, and a carrageenan source. The invention further relates to a method of making a connective tissue analogue, said method comprising preparing a dispersion of a konjac glucomannan source, and a carrageenan source; optionally adding lipid and emulsifying; and functionalizing the konjac glucomannan source and carrageenan source. The invention further relates to a method of making a gelled connective tissue analogue for a food product, said method comprising preparing a dispersion of konjac glucomannan source, a carrageenan source, and monovalent cation salt; optionally adding lipid as oil and / or melted fat to the dispersion and emulsifying; hydrating the konjac glucomannan source and carrageenan source; functionalizing the konjac glucomannan source and carrageenan source; and cooling. The invention further relates to a method of making a gelled connective tissue analogue for a food product, said method comprising a. Preparing a dispersion of konjac glucomannan source, a carrageenan source, and monovalent cation salt in water; b. Optionally adding lipid as oil and / or melted fat to the dispersion, and emulsifying to create an emulsion; c. Hydrating the konjac glucomannan source and carrageenan source by agitating; d. Heating to functionalize the konjac glucomannan source and carrageenan source; and e. Cooling to form a gelled connective tissue analogue. In one embodiment, said method comprises a. Preparing a dispersion of a konjac glucomannan source, a carrageenan source, and a monovalent cation salt in water; b. Optionally adding lipid as oil and / or melted fat to the dispersion, for example sunflower oil, and emulsifying to create an emulsion; c. Hydrating the konjac glucomannan source and carrageenan source by agitating, for example for at least 5 minutes; d. Heating to functionalize the konjac glucomannan source and carrageenan source; and e. Cooling to form a gelled connective tissue analogue. In one embodiment, the carrageenan source comprises over 20% carrageenan, for example between 45% to 55% carrageenan. In one embodiment, the carrageenan source is extracted. In one embodiment, carrageenan is present in the gelled connective tissue analogue at a final concentration between 0.2 wt% to 1.5 wt%. In one embodiment, the monovalent cation salt is present in the gelled connective tissue analogue at a final concentration of between 0.10 wt% to 3.0 wt% sodium chloride or between 0.1 wt% to 0.6 wt% potassium chloride. In one embodiment, the konjac glucomannan source comprises over 50% konjac glucomannan, for example 50 to 75% konjac glucomannan. In one embodiment, the konjac glucomannan source is extracted. In one embodiment, the konjac glucomannan is present in the gelled connective tissue analogue at a final concentration of between 0.3 wt% to 1.5 wt%. In one embodiment, the gelled connective tissue analogue further comprises a protein source. In one embodiment, the protein source is selected from soy protein isolate, gluten isolate, potato protein isolate, and rice protein isolate, preferably soy protein isolate. In one embodiment, the protein source is soy protein isolate added at between 0 to 30 wt%, preferably at about 7.5 wt%. In one embodiment, the dispersion further comprises a starch source. In one embodiment, the starch source is selected from Quinoa flour, Mung Bean starch, Waxy Maize starch, Potato starch, or mixtures thereof. In one embodiment, the starch source is Quinoa flour or Mung Bean starch. In one embodiment, between 0.1 to 10 wt% lipid, preferably about 5 wt% lipid is added in step b). In one embodiment, the food product is a plant based or hybrid food product, for example a ham analogue or steak analogue. The invention further relates to a plant based connective tissue analogue, wherein said analogue comprises about 0.9 wt% konjac glucomannan, about 0.90 wt% carrageenan, about 0.2 wt% KCl, and about 5 wt% oil, wherein the carrageenan source comprises kappa carrageenan and lambda carrageenan in about a 1:1 ratio. The invention further relates to a plant based connective tissue analogue according as described herein, made by a method according to the invention. The invention further relates to a food product comprising the plant based connective tissue analogue of the invention blended with texturized protein. The texturized protein may be wheat gluten and pea protein isolate, for example in the amounts shown in table 1. The texturized protein may be soy protein. The texturized protein may be canola protein. The texturized protein may be a blend of one or more of aforesaid proteins. The invention further relates to the use of plant based connective tissue analogue according to the invention, in a plant based or hybrid food product. Brief description of the figures Figure 1: cohesiveness and juiciness attributes for initial base recipe Figure 2: cohesiveness and juiciness attributes for best variants compared to initial base recipe Figure 3: Parameter 1 of Texture Analysis for DoE18, SF9, SF24 and SF27, compared to initial base recipe Figure 4: Parameter 2 of Texture Analysis for DoE18 and SF27, compared to initial base recipe Figure 5. Friction coefficients as a function of sliding speed (U) measured between glass ball and PDMS surfaces at a tribological force of 0.57 N in presence of Juices extracted via an in vitro simulation of mastication from a ham analogue with egg white as connective tissue, and sample SF27. Error bars indicate standard deviation for a total of six Stribeck curves. Figure 6. Sensory comparison between SF27 and egg white on cohesion and moist perception. 0 baseline = HERTA pork ham reference. Error bars represent the Fischer’s LSD (95% confidence). If the error bars do not overlap, the samples are significantly different in the corresponding sensory attribute. Detailed description Method of making gelled connective tissue analogue A typical gelled connective tissue analogue recipe according to the invention may have a list of ingredients which is substantially the same as the sample IDs listed in Table 6. For example, a gelled connective tissue analogue recipe may have a list of ingredients which are substantially the same as sample IDs SF9, SF23, SF24, SF27 or DoE18. The gelled connective tissue analogue may be made a method according to the invention. For example, the gelled connective tissue analogue may be made by a method which is substantially the same as for sample IDs SF9, SF23, SF24, SF27 or DoE18. The connective tissue analogue may be prepared by dispersing, for example while mixing in a Thermomix, the dry ingredients (konjac glucomannan, kappa-carrageenan and potassium chloride (KCl)) in water. The water may be Vittel water, comprising about 2 mg / kg of potassium. Mixing may be at low speed for about 3 min and then at much higher speed for about 2 sec. High oleic sunflower oil (HOSO) may then be added while mixing at low speed. After complete oil addition, the mixing speed may be increased. For hydration, the speed may be reduced and mixed at low speed for about a further 15 min. The whole process may be done at room temperature, for example following the recipe in Table 3. Water may be mixed with an extruded protein blend. The connective tissue analogue can be added and mixed. The mixture can be placed into a pressure cooker and placed in a steam oven for cooking in order to reach at least 85°C in the core of the mixture. After cooking the ham analogue can be cooled down and sliced. The method may comprise preparing a dispersion of konjac source, carrageenan source, starch source, and KCl, in water, and adding oil, wherein the final concentration of konjac glucomannan, carrageenan, starch, KCl, and oil in the gelled connective tissue analogue is about 0.9 wt% konjac glucomannan, about 0.14 wt% carrageenan, about 5 wt% quinoa flour, about 0.2 wt% KCl, and about 5 wt% oil, wherein the carrageenan is kappa carrageenan. The method may comprise preparing a dispersion of konjac source, carrageenan source, and KCl, in water, and adding oil, wherein the final concentration of konjac glucomannan, carrageenan, KCl, and oil in the gelled connective tissue analogue is about 1.2 wt% konjac glucomannan, about 0.27 wt% carrageenan, about 0.2 wt% KCl, and about 5 wt% oil, wherein the carrageenan is kappa carrageenan. The method may comprise preparing a dispersion of konjac source, carrageenan source, starch source, and KCl, in water, and adding oil, wherein the final concentration of konjac glucomannan, carrageenan, protein source, KCl, and oil in the gelled connective tissue analogue is about 0.9 wt% konjac glucomannan, about 0.14 wt% carrageenan, about 5 wt% soy protein, about 0.2 wt% KCl, and about 5 wt% oil, wherein the carrageenan is kappa carrageenan. The method may comprise preparing a dispersion of konjac source, carrageenan source, and KCl, in water, and adding oil, wherein the final concentration of konjac glucomannan, carrageenan, starch, KCl, and oil in the gelled connective tissue analogue is about 0.9 wt% konjac glucomannan, about 0.90 wt% carrageenan, about 0.2 wt% KCl, and about 5 wt% oil, wherein the carrageenan source comprises kappa carrageenan and lambda carrageenan in about a 1:1 ratio. The method may comprise preparing a dispersion of konjac source, carrageenan source, and KCl, in water, wherein the final concentration of konjac glucomannan, carrageenan, and KCl, in the gelled connective tissue analogue is about 1.1 wt% konjac glucomannan, about 0.25 wt% carrageenan, about 0.3 wt% KCl, wherein the carrageenan is kappa carrageenan. To make a cohesive plant-based ham analogue, the connective tissue analogue is typically used within the following ratio range with plant based extrudate: Plant protein extrudate 70- 80%, connective tissue analogue 20-30%, to a sum of 100.0%. Typically, the connective tissue analogue is used at the following ratio with plant based extrudate and fat analog: Plant protein extrudate 65-75%, Connective tissue analogue 25- 35%, Plant-based fat 3.3%, to a sum of 100.0%. Carrageenan source The carrageenan source may comprise over 20% carrageenan, for example between 45% to 55% carrageenan, or about 95% carrageenan. The carrageenan may comprise kappa carrageenan, or a combination of about 50% kappa carrageenan and about 50% lambda carrageenan. The carrageenan source may be a seaweed flour comprising about 55% carrageenan. Preferably, the seaweed flour is derived from red seaweed. Typically, red seaweed comprises kappa, iota, and lambda carrageenan. Green or brown seaweed are less preferred sources of carrageenan. Typically, carrageenan is present in the gelled connective tissue analogue at a final concentration between 0.2 wt% to 1.5 wt%, or between 0.2 wt% to 1.0 wt%, or between 0.3 wt% to 1.0 wt%, or between 0.4 wt% to 0.9 wt%, or between 0.5 wt% to 0.9 wt%, or between 0.7 wt% to 0.9 wt%, or between 0.8 wt% to 0.9 wt%. Konjac glucomannan source The konjac glucomannan source may comprise over 50% konjac glucomannan, for example between 50 to 75% konjac glucomannan. The konjac source may be a konjac flour comprising between 70 to 75 % glucomannan. The konjac source may be extracted and comprise, for example, about 95.6% glucomannan. Protein source Preferably, the gelled connective tissue analogue further comprises a protein source, for example between 2 to 30%, preferably about 7.5% or about 27% of the protein source. The protein may be added when preparing the dispersion or during the hydration step.. Where a protein isolate is the protein source, then the protein content of the isolate is typically equal or greater than 85% on dry basis, for example about 90% on a dry basis. Where the protein source is wheat gluten, then the protein content is typically equal or greater than 80% on dry basis, for example about 83% on a dry basis. Preferably, the protein source is hydrophobic, for example rice protein isolate. Starch source Preferably, the dispersion further comprises a starch source, preferably up to 10 wt% starch, or about 5 wt% starch. Lipid The addition of lipid as oil and / or melted fat to the dispersion is optional for steak analogues. For ham analogues, the addition of lipid as oil and / or melted fat to the dispersion is preferred. Ham analogue The food product may be a steak or ham analogue comprising the plant based connective tissue analogue blended with texturized protein. When the food product is a ham analogue, then the texturized protein may be the same as that shown in Table 1. The texturized protein may be wheat gluten or pea protein isolate, for example about 12.5 wt% wheat gluten and about 26.5 wt% pea protein isolate. The protein blend may further comprise insoluble particles, for example about 4 wt% insoluble particles. It may further comprise flavors, colors, and preservatives. About 50% water may be added. The total dry matter of the food product may be about 45.4%, or about 41.3% after steam cooking. The ingredient list may be as shown in table 2. Definitions As used herein, the term “about” or “substantially” is understood to refer to numbers in a range of numerals, for example the range of -30% to +30% of the referenced number, or -20% to +20% of the referenced number, or -10% to +10% of the referenced number, or -5% to +5% of the referenced number, or -1% to +1% of the referenced number. All numerical ranges herein should be understood to include all integers, whole or fractions, within the range. As used herein, the term "analogue" is considered to be an edible substitute of a substance in regard to one or more of its major characteristics. As used herein, the term “vegan” refers to an edible composition which is entirely devoid of animal products, or animal derived products, for example eggs, milk, honey, fish, and meat. Those skilled in the art will understand that they can freely combine all features of the present invention disclosed herein. In particular, features described for the compositions of the present invention may be combined with the method or uses of the present invention and vice versa. Further, features described for different embodiments of the present invention may be combined. Where known equivalents exist to specific features, such equivalents are incorporated as if specifically referred to in this specification. Further advantages and features of the present invention are apparent from the figures and non-limiting examples. Examples Example 1 Ham analogue recipe and process The extrudates were assembled with a connective tissue analogue to create a vegan ham loaf. The connective tissue analogue was used to ensure the cohesiveness of extrudate pieces in the final ham analogue. The extrudate used is described in the Table 1 and the vegan ham loaf composition is described in the Table 2. Table 1: extrudate ingredient list Extudate g / 100g Wheat gluten 12.5 Pea protein isolate 26.5 Insoluble particles 4.0 Flavors 3.3 Colors 0.04 Preservative 4.0 Water 50.0 The total dry matter content of the above recipe is 45.4% Table 2: vegan ham loaf ingredient list g / 100g Extrudate pieces 71.0 Added water 4.0 Connective tissue analogue 25.0 The total dry matter content of the above recipe, after steam oven cooking, is 41.3 % The connective tissue analogue was prepared by dispersing, while mixing in a Thermomix, the dry ingredients (konjac glucomannan, kappa-carrageenan and KCl) in Vittel water, containing a fixed amount of 2 mg / kg of potassium, at low speed for 3 min and then at much higher speed 2 sec. The high oleic sunflower oil (HOSO) was then slowly added while mixing at low speed. After complete oil addition, the mixing speed was increased to high speed for 30 sec. For hydration purpose the speed was then reduced and mixed at low speed for a further 15 min. The whole process was done at room temperature and following the recipe in Table 3. Table 3: connective tissue analogue initial base ingredient list Connective tissue analogue initial base recipe g / 100g Vittel Water 93.6 Konjac powder 0.9 Carrageenan 0.3 HOSO 5 KCl 0.2 The total dry matter content of the above recipe is 6.3 %. The added water was manually mixed with the extrudate pieces (see table 2). The connective tissue analogue was then added on top and gently mixed by hand with the wetted extrudates. 750g of the mix (extrudate, water, connective tissue analogue) was inserted into a pressure ham cooker mold (Browin, 1.5kg) comprising a press chamber (dimensions: 168x98mm), a disc plate, a spring and a cover. A wedge (dimensions: 60x90mm) was added on top of the mix to fill the gap in the mold. The mold was then placed in a steam oven for cooking in order to reach 85°C in the core of the vegan ham loaf. After cooking the ham analogue loaf was cooled down overnight in the fridge and then sliced with each slice having a thickness of 2.2mm. Example 2 Optimization of fat analogue binding system performance (juiciness and cohesiveness) The fat analogue carbohydrate-based gel used as a binder (or binding agent) in the cold cut ham analogue comprises a carrageenan source (preferably kappa-carrageenan), a konjac glucomannan source, a mineral salt containing a monovalent cation (preferably potassium from potassium chloride salt), and a liquid oil (preferably high oleic sunflower oil to prevent potential oxidation upon heating and generation of rancid off-flavor). The base recipe used as starting point for the study is described above in example 1. Carrageenan and konjac glucomannan can synergistically form a gel while cooling down and soften or partially melt while heating, which behaves similarly to animal fat. The performance of this initial base carb gel recipe was assessed and optimized. An analytical texture characterization of the generated ham analogue slices prototypes was performed using a cohesiveness measurement method, applied with a TA.HDplusC Texture Analyzer from Stable Micro Systems company. The cohesiveness texture measurement method involved a single penetration of the sliced prototypes, standardized in diameter and thickness (98mm x 2.2mm), held in a circle hole geometry of 63 mm, using a particular ball geometry of 25 mm of diameter. The single penetration enables the generation of graphs and definition of different texture parameters. The aim of this method was to assess 2 different parameters of interest: - Parameter 1: the displacement distance (mm) covered by the geometry from the moment it touches the slice, until the moment the resistance force becomes negligible (threshold set at 10g). The greater the distance, the more cohesive the slice. - Parameter 2: the total work required (force x displacement) to disassemble the analogue ham slices and so negatively impact the cohesiveness. It corresponds to the area under the curve Force (g) x Displacement (mm). The greater the work required, the more cohesive the slice. For each variant, 5 duplicate slices were analyzed to calculate for each defined parameter, the average and the related standard deviations presented in the error bars of the texture analysis graphs. Example 3 Sensory evaluation In parallel, a sensory evaluation of cold-cut pork ham reference slices (thickness 2.2 mm) and prototype slices (thickness 2.2 mm) of the plan described below was performed with trained panelists (n = 13). For this purpose, the following dedicated sensory glossary, focusing specifically on cohesiveness and juiciness attributes, was developed: Table 4: Texture attributes for plant-based ham analogues Attribute Protocol for evaluation Definition name 1- Take each ham slice between the thumb and the middle finger and slightly shake them. 2- Stretch them by pulling on the extremities Capacity of the slice to until breaking them apart. remain cohesive (without any 3- Assess the ham cohesiveness of each sample Cohesiveness cracks, holes or completely in comparison with the reference, considering (Appearance) breaking apart) when it is the following criteria slightly shaken and then -5 → -4 : The slice already breaks apart before stretched with the hands. shaking -4 → -3 : The slice breaks apart while being slightly shaken -3 → -2 : The slice remains cohesive after Attribute Protocol for evaluation Definition name shaking but has clearly less resistance to stretching (e.g. clearly less strength to be applied to break it apart) compared to the reference -2 → -1 : The slice remains cohesive after shaking but has slightly less resistance to stretching (e.g. slightly less strength to be applied to break it apart) compared to the reference -1 → -0 : The slice remains cohesive after shaking with similar resistance to stretching compared to the reference Juiciness Describes how moist the Evaluate the quantity of juice release from the (In-mouth texture feels in the mouth sample along the whole chewing process texture) An in-house panel consisting of thirteen employees was recruited to conduct the sensory comparative profiling of plant-based ham analogues with an animal-based reference (HERTA Le Bon Paris à l’étouffée, France). No selection criteria were used, except for no intolerance to plant-based meat analogues, interest in sensory evaluations and availability. A training session was conducted in a sensory evaluation room. During this session, the panelists were introduced to the texture attributes in the glossary in table 4 and trained using plant-based ham analogues according to the invention and the commercial pork ham reference. The developed sensory assessment method was based on comparative profiling as follows: The panelists were asked to measure the relative intensity of two texture attributes (listed in Table 4) from several plant-based ham analogues compared to the animal-based reference. The linear structured scale for each attribute went from -5 to +5, with the reference sample set at 0. The plant-based ham analogue prototypes under test and the reference were presented at the same time, side by side. As palate cleansing fresh water was used between each assessed sample. The generated sensory results were processed through a statistical analysis to define which samples were significantly different from the reference and from each other, and on which attribute(s). For this purpose, a two-way ANOVA test (sample as fixed and panelist as random factor) treating the data as continuous data (a non-selected attribute was treated equivalent to “not perceived” and assigned as intensity = 0) was applied. As ANOVA indicated significant differences between the samples evaluated in the present study, Fisher’s Least Significant Difference (LSD) was then calculated to determine the significance of the difference between any pair of samples. A 95% confidence level was applied to these statistical tests. The carbohydrate gel cold-set technology has been used to improve the juiciness of the initial non-vegan prototypes for which the fat analogue / binding system used was the animal egg white. The first proposed initial base recipe (described above in example 1) showed a significant improvement of the juiciness, while, nevertheless, the cohesiveness of the slices was negatively impacted (see figure 1). For this reason, a dedicated experimental plan was designed in order to keep the good juiciness level of the initial base recipe, while improving the cohesiveness performance to get closer to the pork ham reference. The experimental plan encompassed: - a DoE approach for the three factors presenting a potential synergy impacting the gel strength (konjac glucomannan, carrageenan, potassium chloride), and - a single factor effect approach where the impact of different recipe and process factors of interest were assessed separately compared to the initial base recipe Table 5: Sample variants of the DoE plan designed on konjac glucomannan, carrageenan and KCl factors Konjac Glucomannan Carrageenan source Potassium Chloride Sample_ID source concentration concentration concentration DoE1 0.80% 0.30% 0.20% DoE2 (Initial Base Recipe) 0.90% 0.30% 0.20% DoE3 1.10% 0.30% 0.20% DoE4 0.80% 0.40% 0.20% DoE5 0.90% 0.40% 0.20% DoE6 1.10% 0.40% 0.20% Konjac Glucomannan Carrageenan source Potassium Chloride Sample_ID source concentration concentration concentration DoE7 0.80% 0.50% 0.20% DoE8 0.90% 0.50% 0.20% DoE9 1.10% 0.50% 0.20% DoE10 0.80% 0.30% 0.30% DoE11 0.90% 0.30% 0.30% DoE12 1.10% 0.30% 0.30% DoE13 0.80% 0.40% 0.30% DoE14 0.90% 0.40% 0.30% DoE15 1.10% 0.40% 0.30% DoE16 0.80% 0.50% 0.30% DoE17 0.90% 0.50% 0.30% DoE18 1.10% 0.50% 0.30% Table 6: Sample variants of the single factors plan KGM Lipid Starch Protein Sample Carrageenan Starch Protein Gel addition source KCl conc source source source ID source conc source source method conc conc conc conc Whole gel SF1 0.90% 0.30% 0% 5% NA NA NA NA recipe added 0% Whole gel SF2 0.90% 0.30% (+ 1% 5% NA NA NA NA recipe NaCl) added Whole gel SF4 0.90% 0.30% 0.20% 0% NA NA NA NA recipe added Whole gel SF5 0.90% 0.30% 0.20% 10% NA NA NA NA recipe added Mung Whole gel Bean SF6 0.90% 0.30% 0.20% 5% 5% NA NA recipe starch added (MBS) Waxy Whole gel Maize SF7 0.90% 0.30% 0.20% 5% 5% NA NA recipe starch added (WMS) Whole gel Potato SF8 0.90% 0.30% 0.20% 5% 5% NA NA recipe starch added KGM Lipid Starch Protein Sample Carrageenan Starch Protein Gel addition source KCl conc source source source ID source conc source source method conc conc conc conc Whole gel Quinoa SF9 0.90% 0.30% 0.20% 5% 5% NA NA recipe flour added 50 / 50 Whole gel SF10 0.90% 0.30% 0.20% 5% WMS 5% NA NA recipe and MBS added Whole gel Best SF11 0.90% 0.30% 0.20% 5% 2.50% NA NA recipe starch added Whole gel Best SF12 0.90% 0.30% 0.20% 5% 7.50% NA NA recipe starch added Whole gel Gluten SF13 0.90% 0.30% 0.20% 5% NA NA 5% recipe isolate added Whole gel Potato SF14 0.90% 0.30% 0.20% 5% NA NA 5% recipe isolate added Whole gel Soy SF15 0.90% 0.30% 0.20% 5% NA NA 5% recipe isolate added Whole gel Rice SF16 0.90% 0.30% 0.20% 5% NA NA 5% recipe isolate added Whole gel Best SF17 0.90% 0.30% 0.20% 5% NA NA 2.50% recipe protein added Whole gel Best SF18 0.90% 0.30% 0.20% 5% NA NA 7.50% recipe protein added Starch / prot powder Soy added SF20A 0.90% 0.30% 0.20% 5% NA NA 26.7% isolate separately to extrudate Starch / prot powder Quinoa added SF20B 0.90% 0.30% 0.20% 5% 26.7% NA NA flour separately to extrudate Whole gel Quinoa Soy SF21 0.90% 0.30% 0.20% 5% 2.5% 3.75% recipe flour isolate added KGM Lipid Starch Protein Sample Carrageenan Starch Protein Gel addition source KCl conc source source source ID source conc source source method conc conc conc conc Starch / prot powder Quinoa Soy added SF22 0.90% 0.30% 0.20% 5% 2.5% 3.75% flour isolate separately to extrudate 1.24% 0.55 Whole gel KGM % SF23 Carrageenan 0.20% 5% NA 0% NA 0% recipe source source n°2 added n°2 Starch / prot powder Soy added SF24 0.90% 0.30% 0.20% 5% NA NA 5% isolate separately to extrudate Mung Whole gel Soy SF25 0.90% 0.30% 0.20% 5% Bean 2.5% 3.75% recipe isolate starch added Starch / prot powder Mung Soy added SF26 0.90% 0.30% 0.20% 5% Bean 2.5% 3.75% isolate separately starch to extrudate 1.52% 1. Whole gel KGM 8% SF27 Carrageenan 0.30% 5% NA 0% NA 0% recipe source source n°2 added n°2 The roll-out of this plan was performed using a KGM source containing 96% glucomannan, and a carrageenan source containing 47% carrageenan (kappa- form exclusively); except for the variants SF23 and SF27, for which other KGM and carrageenan source alternatives were 5 tested, respectively “KGM source n°2” and “Carrageenan source n°2” in the above-mentioned plan. The “KGM source n°2” contains 73% glucomannan, while the “Carrageenan source n°2” contains 49% carrageenan (blend kappa- and lambda- forms, ratio 1:1). All samples of the plan were assessed in Sensory and Texture Analysis applying the methods described above. Sensory data, acquired according to the methodology described herein, were processed with EyeOpenR ® software (Logic 8, Elst, the Netherlands). The “LSD” (Least significant difference) is plotted on the bar charts to show a proxy of the panel intra variability. The confidence level was set to 5 % (p-value < 0.05). LSD bars overlapping signifies the prototypes are not significantly different from each other. Several variants of the plan showed a significative improvement in cohesiveness, while not compromising significantly the juiciness obtained with the initial base recipe (DoE2). Figure 2 shows a comparison in cohesiveness and juiciness assessed in sensory, for the variants DoE2 (Initial base recipe), DoE18, SF9, SF24, and SF27 of the plan, which showed the best results compared to the animal ham reference. In terms of cohesiveness and juiciness, the best variant identified from the DoE part of the plan is the DoE18, consisting in a combination of 1.10% KGM, 0.5% carrageenan, and 0.30% KCl. This variant performs in a similar manner to the “Initial base recipe (DoE2)” in terms of juiciness (LSD bars are overlapping), while showing a clear improvement in terms of cohesiveness (LSD bars are not overlapping). In addition, results of variants SF9 (Quinoa flour as starch source) and SF24 (Soy protein isolate as protein source) clearly show that adding a protein or a starch source in the system also improves the cohesiveness while not negatively impacting the juiciness. For the juiciness attribute, both variants SF9 and SF24 are not significantly different from the “Initial base recipe (DoE2)” (LSD bars are overlapping for the 2). Nevertheless, in terms of cohesiveness, the variant SF9 shows an improvement which is not statistically significant (LSD bars are overlapping with the initial base recipe), while SF24 shows a significant improvement (LSD bars are not overlapping with the initial base recipe). Finally, variant SF27 (use of other KGM and carrageenan source alternatives, with highest concentrations) shows the best results for both juiciness and cohesiveness attributes. In terms of juiciness, this variant performs in a similar manner to the “Initial base recipe (DoE2)” (LSD bars are overlapping), but also to the animal pork ham reference (LSD bar is crossing the “0” line which corresponds to the sensory reference benchmark). In terms of cohesiveness, this variant shows the best result, which is significantly improved compared to the “Initial base recipe (DoE2)” (LSD bars are not overlapping) and very close to 0 which corresponds to the performance of the animal pork ham reference. A combination of the best DoE recipe, using the KGM source n°2 and carrageenan source n°2, and the single factor “soy protein isolate” is the most promising compromise to tackle the cohesiveness and juiciness at the same time for vegan ham analogue slices application. Example 4 Texture analysis To confirm the above results, the texture analysis parameter 1 described above was used and results are shown in Figure 3, for the variants DoE18, SF9, SF24 and SF27 compared to DoE2 (Initial base recipe). The texture analysis data for the parameter 1 allow to draw the same conclusions than the sensory data. The cohesiveness of DoE18, SF24 and SF27 is significantly improved compared to the initial base recipe (DoE2), while the cohesiveness improvement for the SF9 variant is not statistically significant. Indeed, for the texture analysis parameter 1, the error bars of DoE18, SF24 and SF27 are not overlapping with the one of the initial base recipe (DoE2), while the error bar of SF9 is overlapping. The other texture analysis (parameter 2) described above allows to draw the same conclusions for the best variant from the DoE part of the plan (DoE18) and the best variant from the single factor part of the plan (SF27). The results are shown in Figure 4, for the variants DoE18 and SF27 compared to DoE2 (Initial base recipe). The texture analysis data for the parameter 2 allow to draw the same conclusions than the sensory data. The cohesiveness of DoE18 and SF27 are significantly improved compared to the initial base recipe (DoE2). Indeed, for the texture analysis parameter 2, the error bars of DoE18 and SF27 are not overlapping with the one of the initial base recipe (DoE2). In conclusion, looking at sensory and texture analysis data, the variant SF27 clearly shows the best results in terms of cohesiveness. Also, the sensory data confirm that the SF27 variant provides the best performance in terms of juiciness. Example 5 Evaluation of the fat analogue binding system performance using Tribology To confirm the above results, tribology measurements on meat analogue Juices were performed. Juices were extracted from the ham analogues via an in vitro simulation of mastication: slices of ham were heated at 40°C and ground in a Grindomix mixer for 5 seconds. The ground mass was mixed with 40 °C Vittel water in a Stomacker bag (Nasco, Whirl-Pak, Madison USA) for 30 seconds inside a Stomacher mixer (Laboratory blenders stomacher 400, Seward) at a final ratio of 1:4. The juice was extracted for 2 minutes from the mass using a 100 µm mesh filtering unit (Steriflip 50 mL, Merck Millipore). The tribological properties of the juices were investigated using a MCR rheometer (MCR 702, Anton Paar, Austria) with a tribology-cell attachment (measuring shaft BC 12.7), using a ball- on-three-pin test configuration. The ball is of soda-lime glass with a diameter of 12.7 mm, while the pins are of polydimethylsiloxane (PDMS) with a diameter of 6 mm, a height of 6 mm, an E-modulus of 5000 kPa, a Poison’s ratio of 0.40, and a surface roughness of 0.17 µm. The friction coefficients were measured at logarithmically increasing rotation speeds from 1x10-5to 0.2 m / s at a tribological force of 0.57 N and a temperature of 37°C, similar to in- mouth temperature. Results are shown in Figure 5, for the variants SF27 compared to a ham analogue made with egg white as connective tissue analogue (commercial product). Notably, the friction data shows that the egg white binder produces juices with a higher friction compared to sample SF27. These results indicate that the addition of the connective tissue analogue brings lubrication properties to juices. Such juices are expelled from the matrix during the mastication of the ham analogue and coat the oral cavity, which increases the moist perception of the analogue while maintaining cohesion, as showed in Figure 6.

Claims

CLAIMS 1. A method of making a gelled connective tissue analogue for a food product, said method comprising a. Preparing a dispersion of a konjac glucomannan source, a carrageenan source, and a monovalent cation salt in water; b. Optionally adding lipid as oil and / or melted fat to the dispersion, for example sunflower oil, and emulsifying to create an emulsion; c. Hydrating the konjac glucomannan source and carrageenan source by agitating, for example for at least 5 minutes; d. Heating to functionalize the konjac glucomannan source and carrageenan source; and e. Cooling to form a gelled connective tissue analogue, wherein the carrageenan source comprises over 20% carrageenan.

2. The method according to claim 1, wherein the carrageenan source comprises between 45% to 55% carrageenan.

3. The method according to any one of claims 1 or 2, wherein the carrageenan source is extracted.

4. The method according to any one of claims 1 to 3, wherein carrageenan is present in the gelled connective tissue analogue at a final concentration between 0.2 wt% to 1.5 wt%.

5. The method according to any one of claims 1 to 4, wherein the monovalent cation salt is present in the gelled connective tissue analogue at a final concentration of between 0.10 wt% to 3.0 wt% sodium chloride or between 0.1 wt% to 0.6 wt% potassium chloride.

6. The method according to any one of claims 1 to 5, wherein the konjac glucomannan source comprises over 50% konjac glucomannan, for example 50 to 75% konjac glucomannan.

7. The method according to any one of claims 1 to 6, wherein the konjac glucomannan source is extracted.

8. The method according to claims 1 to 7, wherein the konjac glucomannan is present in the gelled connective tissue analogue at a final concentration of between 0.3 wt% to 1.5 wt%.

9. The method according to any one of claims 1 to 8, wherein the gelled connective tissue analogue further comprises a protein source.

10. The method according to claim 9, wherein the protein source is selected from soy protein isolate, gluten isolate, potato protein isolate, and rice protein isolate, preferably soy protein isolate.

11. The method according to any one of claims 9 or 10, wherein the protein source is soy protein isolate added at between 0 to 30 wt%, preferably at about 7.5 wt%.

12. The method according to any one of claims 1 to 11, wherein the dispersion further comprises a starch source.

13. The method according to claim 12, wherein the starch source is selected from Quinoa flour, Mung Bean starch, Waxy Maize starch, Potato starch, or mixtures thereof.

14. The method according to any one of claims 12 or 13, wherein the starch source is Quinoa flour or Mung Bean starch.

15. The method according to any one of claims 1 to 14, wherein between 0.1 to 10 wt% lipid, preferably about 5 wt% lipid is added in step b).

16. The method according to any one of claims 1 to 15, wherein the food product is a plant based or hybrid food product, for example a ham analogue or steak analogue.

17. A plant based connective tissue analogue, wherein said analogue comprises from 0.3 wt% to 1.5 wt% konjac glucomannan, from 0.2 wt% to 1.5 wt% carrageenan,from 0.1 wt% to 0.6 wt% potassium chloride, and from 0.1 wt% to 10 wt% oil, wherein the carrageenan source comprises kappa carrageenan and lambda carrageenan in about a 1:1 ratio.

18. A plant based connective tissue analogue, wherein said analogue comprises about 0.9 wt% konjac glucomannan, about 0.90 wt% carrageenan, about 0.2 wt% KCl, and about 5 wt% oil, wherein the carrageenan source comprises kappa carrageenan and lambda carrageenan in about a 1:1 ratio.

19. A plant based connective tissue analogue according to claim 17 or 18, made by a method according to any one of claims 1 to 16.

20. A food product comprising the plant based connective tissue analogue according to any of claims 17 to 19 blended with texturized protein.

21. Use of plant based connective tissue analogue according to claim 17, in a plant based or hybrid food product.

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