Technical field

The invention relates to a method of thickening and texturizing fruit and vegetable-based purees and products thereof.

Background

Current consumer trends are leading to increased demand of clean-label plant-based products. This is challenging the food industry to research clean-label ingredients and technologies that enable enough thickening and texturization of plant-based products, which have an exciting texture and a healthy appearance. To meet these consumer demands, a continuous improvement of the product portfolio is required to replace existing binders, such as egg white and methylcellulose.

Summary of invention

The invention relates to a vegan binder that is based on the thickening and texturization of fruit and vegetable purees and products thereof. Such a binding technology improves the nutritional profile and provides a vegan based binder for products targeting infants and toddlers. Moreover, nutritional trends are directed towards healthier products, and improving current products on the market which are not meeting consumer needs.

It has surprisingly been shown that fruit and vegetable purees, as well as pectin-containing fibers, can be thickened and texturized using a plant-derived pectin methylesterase- containing enzyme preparation, like papain. Moreover, texturized fruit and vegetable purees which are enriched with a calcium source and fiber, such as citrus fiber, followed by an incubation with papain, can be used as a binder for various product concepts. Examples of the latter are patties, burgers, rolls, sausages, vegetable and fruit puree-based snacks in various forms and shapes, freeze dried snacks, baby and infant purees, chocolate fillings.

The invention relates in general to a method of making a puree product, said method comprising mixing a puree with a pectin methylesterase.

The invention further relates to a method of making a puree product, said method comprising forming a puree mixture by mixing fruit and/or vegetable puree with a plant-based pectin methylesterase.

The invention further relates to a method of making a puree product, said method comprising forming a puree mixture by mixing fruit and/or vegetable puree with a plant-based pectin methylesterase; optionally molding the puree mixture; incubating the puree mixture; and heat treating the puree mixture to inactivate the pectin methylesterase.

The invention further relates to a method of making a thickened or texturized puree product, said method comprising a. Forming a puree mixture by mixing fruit and/or vegetable puree with a plant-based pectin methylesterase; b. Optionally molding the puree mixture; c. Incubating the puree mixture; and d. Heat treating the puree mixture to inactivate the pectin methylesterase.

In one embodiment, the puree mixture comprises between 0.1 to 4 wt%, or between 0.4 to 3 wt%, or between 1 to 2 wt%, or about 1.5 wt% of a plant-based pectin methylesterase.

In one embodiment, the plant-based pectin methylesterase is from a papaya, kiwi, or pineapple source. Preferably, the plant-based pectin methylesterase is papain from papaya.

In one embodiment, the puree mixture further comprises up to 0.01 to 5 wt%, or between 1 to 3 wt%, of a calcium source.

In one embodiment, the calcium source is selected from calcium chloride, Ca-lactate 4.5- hydrate (calcium L-lactate hydrate), or calcium from a marine algae source, preferably calcium chloride.

In one embodiment, the calcium source is selected from calcium chloride, calcium lactate, or calcium from a marine algae source, preferably calcium chloride.

In one embodiment, the puree mixture further comprises up to 4 wt% added fiber, or between 0.01 to 3.5 wt%, or between 1 to 2 wt% added fiber, wherein said fiber has been isolated from an apple or citrus fruit source.

In one embodiment, the fruit and/or vegetable puree has a pH of between 3.0 to 7.0, preferably between 3.8 to 5.8.

In one embodiment, the puree mixture comprises between 20 to 99.5 wt% fruit and/or vegetable puree.

In one embodiment, the incubation time in step c is between 1 to 120 minutes, or about 120 minutes.

In one embodiment, the fruit and/or vegetable puree is from carrot, apple, parsnip, mango, pear, or butternut squash.

The invention further relates to a method of making a texturized puree product, said method comprising forming a puree mixture by mixing fruit and/or vegetable puree with a preparation comprising plant-based pectin methylesterase; placing the puree mixture in a mold; incubating the puree mixture in the mold to obtain a texturized puree product; and heat treating the texturized puree product to inactivate the pectin methylesterase.

In one embodiment, said method comprises forming a puree mixture by mixing fruit and/or vegetable puree with a preparation comprising plant-based pectin methylesterase prepared from a papaya, kiwi, or pineapple source, preferably a papain preparation from a papaya plant; placing the puree mixture in a mold; incubating the puree mixture in the mold to obtain a texturized puree product; and heat treating the texturized puree product to at least 70°C to inactivate the pectin methylesterase.

In one embodiment, the puree mixture comprises between 0.1 to 4 wt% of a preparation comprising plant-based pectin methylesterase prepared from a papaya, kiwi, or pineapple source, preferably papain from papaya. In one embodiment, the fruit and/or vegetable puree is mixed with a preparation comprising plant-based pectin methylesterase prepared from a papaya plant.

In one embodiment, the papain preparation has a protein concentration of between 30 to 93 wt%.

In one embodiment, the puree mixture further comprises up to 0.01 to 5 wt% of a calcium salt, wherein said calcium salt does not originate from a fruit and/or vegetable source.

In one embodiment, the calcium source is selected from calcium chloride, calcium lactate, Ca- lactate 4.5-hydrate (calcium L-lactate hydrate), or calcium from a marine algae source, preferably calcium chloride.

In one embodiment, the puree mixture further comprises up to 4 wt% added fiber, wherein said fiber has been isolated from an apple or citrus fruit source. In one embodiment, the added fiber is a fiber preparation comprising at least 25 wt% fiber.

In one embodiment, the fruit and/or vegetable puree has a pH of between 3.0 to 7.0, preferably between 3.8 to 5.8.

In one embodiment, the puree mixture comprises between 20 to 99.5 wt% fruit and/or vegetable puree.

In one embodiment, the incubation time is between 1 to 120 minutes to obtain a texturized puree product.

In one embodiment, the fruit and/or vegetable puree is from carrot, apple, parsnip, mango, pear, or butternut squash.

The invention further relates to a thickened or texturized puree product made by a method according to the invention.

In one embodiment, the invention relates to a texturized puree product made by a method according to the invention.

In one embodiment, the product is heat stable, for example at above 90°C.

In one embodiment, the product is cold stable, for example at below -18°C.

In one embodiment, the texturized puree product comprises less than 1 wt% calcium salt.

In one embodiment, the puree product comprises calcium and pectin fiber, wherein the pectin fiber has a degree of methylation below 50%.

In one embodiment, the product is a thickened puree product with less than 1 wt% calcium.

The invention further relates to a food product comprising at least 20 wt% of the thickened or texturized puree product according to the invention.

The invention further relates to a food product, wherein said food product comprises between 5 to 80 % of the texturized puree product according to the invention.

In one embodiment, said food product is freeze dried. The invention further relates to the use of a plant-based pectin methylesterase to make a thickened or texturized fruit and/or vegetable puree product as described herein.

The invention further relates to the use of a plant-based pectin methylesterase from a papaya, kiwi, or pineapple source, preferably papain from papaya, to make a texturized fruit and/or vegetable puree product as described herein.

Detailed description of the invention

The method of making a thickened or texturized puree product is described below. A puree mixture is formed by mixing fruit and/or vegetable puree with enzyme.

A pectin methylesterase-containing enzyme preparations can then be added at a concentration ranging from between 0.01 to 4% (w/w). The pethin methylesterase enzyme may be in powder or liquid form. Preferably, the puree mixture is cooled before addition of pectin methylesterase, for example to less than 6°C, or to between 4 to 6°C.

The enzyme may originate from plants or from fungi, preferably from plants, such as papain.

A divalent metal ion source, preferably a calcium source such as calcium chloride can be added to the puree mixture. The divalent metal ion source can be added at a concentration between 0.01 to 5% (w/w).

The divalent metal ion source can be added before the puree cooking or prior to incubation of the puree with a pectin methylesterase-containing enzyme.

Preferably, the calcium source is selected from calcium chloride, Ca-lactate 4.5-hydrate (calcium L-lactate hydrate), or calcium from a marine algae source, preferably calcium chloride.

A fiber source, preferably a citrus fiber, can be added to the puree mixture. The fiber source can be added at a concentration between 0.01 to 5 wt%, or between 0.01 to 4 wt%. The fiber source is preferably a concentrated or an isolated fiber source, for example isolated from apple or citrus fruit.

The pH of the puree mixture can be between or adjusted to between pH 3.0 to 7.0, or below pH 5.0, to between 3.8 to 4.8, orto between 4.7 to 4.95. The pH can be adjusted, for example, by using citric acid or lemon juice.

The puree mixture may comprise between 20 to 99.5 wt%, or between 30 to 99.5 wt%, or between 40 to 99.5 wt%, or between 50 to 99.5 wt%, or between 60 to 99.5 wt%, or between 70 to 99.5 wt%, or preferably between 80 to 99.5 wt% fruit and/or vegetable puree.

The fruit and/or vegetable puree can be, for example, from carrot, apple, parsnip, mango, pear, or butternut squash.

After mixing the divalent metal ion source and fiber source in the puree mixture, the puree mixture can be cooled to less than 6°C, for example to between 4 to 6°C.

The puree mixture can be further mixed and filled into molds. The enzyme incubation can be performed at about 50°C for about 1 hour. The puree mixture can be cooked, for example until a core temperature of at 80°C for at least 3 minutes is achieved. The cooker can be set at about 100°C, for example for about 20 minutes. After cooking, the puree mixture can be mixed preferably until it is homogenous. The mixtures can then be stored at between 4 to 6°C. Longer term storage, for example more than 24 hours, can be in the freezer.

The thickened or texturized puree product can be vegan or vegetarian.

The puree mixture can be made from parsnip. The puree mixture can be made from kiwi. The puree mixture can be made from a combination of parsnip and kiwi. The puree mixture can be made from butternut squash. The puree mixture can be made from a combination of butternut squash and parsnip. The puree mixture can be made from apple. The puree mixture can be made from mango. The puree mixture can be made from a combination of apple and banana. The puree mixture can be made from a combination of apple, strawberry, raspberry, banana, and carrot juice.

Calcium chloride can be added to the puree mixture, for example at about 0.375 % (w/w), or between 0.4 to 1.5 % (w/w), or between 0.5 to 1.5 % (w/w). Citrus fiber can be added to the puree mixture. The puree mixture may comprise at least 0.6 % (w/w) calcium source, or between 0.7 to 1.5 % (w/w) calcium source, or between 0.8 to 1.2 % (w/w) calcium source, for example calcium chloride.

The puree mixture may comprise sodium chloride, for example between 0.01 to 0.05 g/lOOg sodium chloride.

The puree mixture may comprise at least 0.1 % (w/w) pectin methylesterase, or between 0.1 to 1 % (w/w), or between 0.1 to 0.5 % (w/w) pectin methylesterase, for example papain. The puree mixture may comprise a combination of fungal pectin methylesterase and papain.

The calcium source can be calcium chloride, Ca-lactate 4.5-hydrate (calcium L-lactate hydrate), or a marine algae source. Preferably, the calcium source is calcium chloride, for example calcium chloride dihydrate. The marine algae source may be a calcium-containing preparation from the calcareous marine algae comprising 20 - 40% calcium ions. The source may comprise, for example, between 30 to 32 wt% calcium ions.

The procedure to obtain the purees may be substantially as shown in Table 1.

The recipe may comprise the same ingredients as a recipe described in the examples section, such as Examples 26, 27, 33, 34, 35, 43, 46, 47, 51, 55, 59, 75, 85, 88, 96, 103, 117, 137, 138, 165, 181, 184, 189, 200, 210, 229, 236, 237, and 250.

The recipe may comprise any of the following combinations of ingredients (% values can vary by up to 30% compared to those shown):

Parsnip, Water, Mango, Lemon Juice, [Calcium chloride dihydrate], and papain, for example at about the following concentrations: Parsnip 57,8%, Water 25%, Mango 14%, Lemon Juice 2,2%, 0,8% [Calcium chloride dihydrate], and 0,4% Papain; or

Parsnip, Water, Blueberry, Lemon Juice, [Calcium chloride dihydrate], and papain, for example at about the following concentrations: Parsnip 61,6%, Water 27%, Blueberry 8%, Lemon Juice 2,4%, 0,8% [Calcium chloride dihydrate], and 0,4% Papain; or

Pears (incl. peel), [Calcium chloride dihydrate], and Papain, for example at about the following concentrations: Pears (incl. peel) 98,8%, 0,8% [Calcium chloride dihydrate], and 0,4% Papain; or Mango (only flesh), [Calcium chloride dihydrate], and Papain, for example at about the following concentrations: Mango (only flesh) 98.8%, 0.8% [Calcium chloride dihydrate], and 0.4% Papain; or

Apple - Granny Smith (incl. peel), [Calcium chloride dihydrate], and Papain, for example at about the following concentrations: Apple - Granny Smith (incl. peel) 98,8%, 0,8% [Calcium chloride dihydrate], and 0,4% Papain; or

Apple, Banana, [Calcium chloride dihydrate], and Papain for example at about the following concentrations: 70% Apple, 30% Banana (TS at about 17.5%), 0,6% [Calcium chloride dihydrate], and 0,4% Papain; or

Apple, strawberry, raspberry and banana, carrot juice, [Calcium chloride dihydrate], and Papain, for example at about the following concentrations: Apple 77%, strawberry 10%, raspberry and bananas 4%, carrot juice (TS at about 15.8%), 0,6% [Calcium chloride dihydrate], and 0,4% Papain; or

Rice flour, banana, pear, papain, calcium chloride, and lemon juice, for example at about the following concentrations: Rice flour 5.1, banana 60.0, pear 32.9, papain 0.4, calcium chloride 0.8, and lemon juice 0.8; or

Rice flour, banana, apple, blueberry powder, papain, calcium chloride, and lemon juice, for example at about the following concentrations: Rice flour 5.1, banana 60.0, apple 31.9, blueberry powder 1.0, papain 0.4, and calcium chloride 0.8; or

Carrot puree, citrus fiber, calcium chloride, papain, sugar beet flakes, for example at about the following concentrations: 91.5 to 97 Carrot puree, 0.5 to 2.0 citrus fiber, 0.5 to 2.0 calcium chloride, 0.5 to 2.0 papain, 2.5 sugar beet flakes; or

Chicory puree, citrus fiber, calcium chloride, papain, sugar beet flakes, for example at about the following concentrations: 91.5 to 97 Chicory puree, 0.5 to 2.0 citrus fiber, 0.5 to 2.0 calcium chloride, 0.5 to 2.0 papain, 2.5 sugar beet flakes; or

Carrot puree, Chicory puree, citrus fiber, calcium chloride, papain, sugar beet flakes, for example at about the following concentrations: 55.1 to 57.8 carrot puree, 36.4 to 38.2 Chicory puree, 0.5 to 2.0 citrus fiber, 0.5 to 2.0 calcium chloride, 0.5 to 2.0 papain, 2.5 sugar beet flakes.

Preferably, the puree product comprises less than 1 wt% calcium source, for example calcium chloride. Products comprising less than 1 wt% calcium source are suitable for consumption by infants.

The puree product can have about the following nutrient composition (in g/lOOg) before freeze drying of about 99% puree: 0.1 Fat, 11.3 Carbohydrates, 10.6 sugar, 0.4 protein, 0.05 salt, 0.02 sodium, 0.6 papain, and 0.4 calcium chloride.

The puree product can have about the following nutrient composition (in g/lOOg) after freeze drying puree wherein the total solids is at least 95%: 0.5 Fat, 54.2 Carbohydrates, 50.9 sugar, 1.9 protein, 0.2 salt, 0.1 sodium, 2.9 papain, and 1.9 calcium chloride.

Nutritional values can vary by up to 30% compared to those shown.

The invention further relates to a food product comprising a thickened or texturized fruit and/or vegetable puree. The food product may be a vegetable roll, a plant-based patty, a filling. The plant based patty may comprise, for example chickpea, brown rice, vegetables, and herbs.

The filling may be, for example, a chocolate filling.

Preferably, the food product comprises up to 10 wt%, or up to 15 wt%, or up to 20 wt% of the thickened or texturized fruit and/or vegetable puree.

The food product can be vegan or vegetarian.

As used herein, the term "about" 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.

As used herein, the term "vegetarian" relates to an edible composition which is entirely devoid of meat, poultry, game, fish, shellfish or by-products of animal slaughter.

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

Method of making fruit and vegetable purees

Fresh fruit and vegetable purees were prepared using a Thermomix (Vorwerk Thermomix TM6 or TM31). The fruit and vegetables were ordered from a local vendor. First, fruit and vegetables were washed, cleaned, peeled, and cut priorto using the Thermomix. Purees were weighed in a range between 600 to 850 g (Table 1). If the pH of the fruit and vegetables was above 5.0, either citric acid or lemon juice was added to ensure a pH below 5.0 during the heating step. To determine the required amount of citric acid or lemon juice, 50 g of a fruit or vegetable paste was prepared and diluted in 50 mL MQ water. Subsequently, the pH was adjusted to 4.7 to 4.95 and the determined amount of acidifierwas used to calculate the citric acid or lemon juice amount that was needed to set the pH below 5.0 for 600 to 800 g of fresh fruit and vegetables (Table 1). A source of a divalent metal ion (calcium derivatives, such as calcium chloride) ranging from 0.01 to 5% (w/w) was added either before the puree cooking or prior to conduction of the experiments which involved the incubation of the prepared purees with a pectin methylesterase-containing enzyme preparation originating from plants, such as papain, or fungi.

Commercial fruit and vegetables purees were purchased from a local vendor. If the pH of the fruit and vegetables was above 5.0, either citric acid or lemon juice was added to ensure a pH below 5.0 during the heating step. To determine the required amount of citric acid or lemon juice, 50 g of a fruit or vegetable paste was prepared and diluted in 50 mL MQ water. Subsequently, the pH was adjusted to 4.7 to 4.95 and the determined amount of acidifier was used to calculate the citric acid or lemon juice amount that was needed to set the pH below 5.0. A source of a divalent metal ion (calcium derivatives, such as calcium chloride) ranging from 0.01 to 5% (w/w) was added prior to conduction of the experiments which involved the incubation of the purees with a pectin methylesterase-containing enzyme preparation.

Table 1: Procedure to obtain homogenous fruit and vegetable purees using a Thermomix

Example 2

Method of making a thickened or texturized fruit and vegetable puree using fungal and plant pectin methylesterases

To determine the texturization effect of pectin methylesterase-containing enzyme preparations on fruit and vegetable purees, a bench scale setup was developed. Therefore, 50 mL falcon tubes, a drilling machine with propeller for mixing of the samples and a water bath was used. In general, 20 to 25 g of fruit and vegetable puree was weighed in and cooled (4 - 6°C). After cooling, the pectin methylesterase-containing enzyme preparations, which were either powder or liquid, were added to the puree. After rigorous mixing of the samples for 30 seconds, using a drilling machine and a tailor-made stirring rod, samples were incubated in a water bath. The enzyme reaction was stopped by placing the incubates into a water bath that was pre-set to a temperature of 90°C for at least 10 min. The texture of the samples was evaluated visually, by placing the samples on a plastic rack, or by using a texture analyzer. Depending on the type of experiment, parameters varied, such as the pectin methylesterase-containing enzyme preparation source (i.e. fungal or plant), commercial provider of the enzyme, different enzyme amounts, different calcium amounts, alternative calcium sources, or addition of pectic material (i.e. citrus fiber). An overview of the general process flow for the texturization of fruit and vegetable purees using pectin methylesterase- containing enzyme preparations is shown in Figure 1.

Determination of the texture (TA Analysis)

Texture profile analysis was conducted using a TA XT Plus Texture Analyser (Stable Micro Systems, UK). Puree samples (weight 100 ± 1 g) were loaded in a cylindric beaker (49 mm diameter x 60 mm height) and tapped to level the surface. A compression test was performed using a P/60C acrylic cylindrical probe at a speed of 2 mm sec ^-1 up to a distance of 30 mm. All measurements were made in triplicate. The software "Exponent" was used to extract data from the curve and exported to Excel. Texturized purees or fibers which were prepared in a 50 mL falcon tube were measured using the Zwick Roell Z005texture analyzer (n° 3062814) which was equipped with a cylindrical probe (6 mm diameter). The penetration depth was 25 mm and the speed was set to 0.5mm/second. The analysis was performed at room temperature (20-22°C).

Determination of the viscosity (Bostwick Viscosimeter Consistometer)

A standard Bostwick consistometer (Greensenselab, UK) was used. To perform a test, 100 g of puree was loaded into the compartment and leveled with a metal spatula. Then the gate was opened, and a stopwatch was started simultaneously. At a predetermined time (0, 5, 10, 15, 20, 30, 45, 60, 75 and 90 seconds), the position of the liquid in the trough was recorded using a camera directly held over the consistometer. In this work, the gate was opened when the liquid's temperature inside the compartment reached room temperature (21 ± 0.5C). If applicable (carrot puree), maximum reading at the center of the trough and minimum reading at the edges of the trough were recorded then averaged. Each product was measured twice or triple and averaged. The Bostwick consistometer was washed thoroughly in warm soapy water and rinsed under running water for at least 1 minute then was dried completely with paper towels and a stream of air between each trial.

Examples 3

Method of making a shaped fruit and vegetable puree using fungal and plant pectin methylesterase-containing enzyme preparations

Larger amounts of fruit and vegetable purees were needed to prepare puree based experiments. In general, the enzymatic process was based on the above-described process. Purees were weighed in kitchen bowls. The agitation was performed using a spoon or whisk. The calcium source (ranging from 0.01 to 5% (w/w)) and, if needed, fiber (citrus fiber, ranging from 0.01 to 5% (w/w)) was mixed with the puree and the whole preparation was cooled down to 4 - 6°C. After cooling, the pectin methylesterase-containing enzyme preparations (ranging from 0.01 to 4% (w/w)), which were either powder or liquid, were added to the puree. After rigorous mixing of the samples for 30 seconds, the cooled preparation was immediately filled in molds (i.e. bear shape). Afterwards, the molds were covered with a plastic foil. The enzyme reaction was performed at 50°C for 1 h using a kitchen oven. Afterwards, the temperature of the oven was increased to 100°C and the samples were heated until a core temperature of 80°C for 3 minutes was achieved. As a next step, samples were cooled down to room temperature and placed in a fridge, followed by demolding after reaching a temperature below 10°C (shaped puree products).

Alternatively, after demolding, the shaped purees were frozen and freeze-dried (shaped and freeze-dried puree products). An overview of the general process flow for the texturization (including molding) of fruit and vegetable purees pectin methylesterase-containing enzyme preparations (kitchen scale experiments) is shown in Figure 2.

Examples 4 - 11

Method of making a texturized parsnip puree using papain and a kiwi extract

To determine the texturization effect of pectin methylesterase-containing enzyme preparations papain and a kiwi extract on fruit and vegetable purees, a bench scale setup was used. Therefore, 25 g of fruit and vegetable puree was weight in 50 mL falcon tubes and cooled (4 - 6°C). After cooling, calcium chloride (0.375 %(w/w)) and, if applicable, citrus fiber (1.5 %(w/w)) was added. Samples were incubated with and without the enzyme preparations, which were either powder or liquid, for 1 h at 50°C. The enzyme reaction was stopped by placing the incubates into a water bath which was pre-set to a temperature of 90°C for at least 10 min. The texturization of the samples was determined by the texture analyzer. The required forces are summarized in Table 2. In general, purees incubated with the enzyme preparations in the presence or absence of citrus fiber led to the formation of firmer gels compared to purees that were incubated without the addition of plant preparations. Both the kiwi extract and papain led to the formation of texturized purees after incubation compared to the samples incubated without enzyme addition.

Table 2: Experimental setup and texture analysis results of the incubation of parsnip puree with the pectin methylesterase-containing enzyme preparations papain and a kiwi extract

Examples 12 - 35

Method of making a texturized parsnip puree using two papain concentrations and an increasing calcium amount

To determine the texturization of parsnip puree incubated with papain (0.1 and 0.4 % (w/w)) in the presence of increasing calcium concentrations (0 - 1.2 %(w/w)), a bench scale setup was used. The texturization of the samples was determined by the texture analyzer. The required forces are summarized in Table 3. In general, an increase in the calcium concentration led to an enhanced gel formation if purees were incubated with either 0.1 or 0.4 % (w/w) papain. The addition of calcium chloride did not lead to a gel formation if purees were incubated without papain. To achieve a significant increase in puree texture, a calcium concentration of at least 0.4 (w/w) and 0.8 % (w/w) was needed, if 0.1 or 0.4 % (w/w) of papain was added, respectively.

Table 3: Experimental setup and texture analysis results to determine the texturization of parsnip puree incubated with papain (0.1 and 0.4 % (w/w)) using increasing calcium concentrations (0 - 1.2% (w/w)).

Examples 36 - 55

Method of making a texturized butternut squash puree using two papain concentrations and an increasing calcium amount

To determine the texturization of butternut squash puree incubated with papain (0.1 and 0.4 % (w/w)) in the presence of increasing calcium concentrations (0 - 1.2 %(w/w)), a bench scale setup was used. The texturization of the samples was determined by the texture analyzer. The required forces are summarized in Table 4. In general, an increase in the calcium concentration increased the texture if purees were incubated with either 0.1 or 0.4 % (w/w) papain. The addition of calcium chloride did not lead to a gel formation if the butternut squash purees were incubated without papain. To achieve a significant increase in puree texture, a calcium concentration of at least 0.4 (w/w) and 0.8 % (w/w) was needed, if 0.1 or 0.4 % (w/w) of papain was added, respectively.

Table 4: Experimental setup and texture analysis results to determine the texturization of butternut squash puree incubated with papain (0.1 and 0.4 % (w/w)) using increasing calcium concentrations (0 - 1.2% (w/w))

Examples 56 - 83

Method of making a texturized commercial puree using two papain concentrations and various calcium salts

To determine the effect of various calcium salts (0.8 %(w/w)) on the texturization of a commercial fruit puree (Apple 77%, Strawberry 10%, Raspberry + Banana 4%, Carrot juice (pH 3.72; TS 15.83%)) incubated with papain (0.1 and 0.4 % (w/w)), a bench scale setup was used. Calcium chloride was used as a benchmark and supplemented with an equal molar calcium ion ratio using various salt alternatives. An overview of the seven calcium salts, concentration and experimental setup is presented in Table 5. The texturization of the samples was determined by the texture analyzer and required forces are summarized in Table 5, as well.

In general, the strongest texture (Force 0.6 - 1.4 N) of a commercial fruit puree incubated with papain was achieved in the presence of calcium chloride. Promising calcium chloride alternatives are Ca-lactate 4.5-hydrate (calcium L-lactate hydrate), marine algae source 1 and 2, as the required force to penetrate the texturized purees after the incubation with papain was similar (Force 0.2 - 1.4 N). Purees incubated with a higher amount of papain (0.4% (w/w)) led to a stronger texturization compared to purees incubated with a lower amount of papain (0.1% (w/w)). Notably, an increase of the incubation time from 1 to 2 h did not lead to an increased texturization of purees which were incubated with 0.4% (w/w) papain.

Table 5: Results of the texture analysis using calcium alternatives

Examples 84 - 99

Method of making a texturized butternut squash puree and parsnip puree incubated with either a fungal or plant pectin methylesterase-containing enzyme preparation (Papain,

Bromelain)

The effect of a fungal (derived from Aspergillus species) and two plant-derived pectin methylesterase-containing enzyme preparations (Papain, Bromelain) on the texturization of butternut squash and parsnip puree in the presence of calcium chloride (0.4 %(w/w)) was visually determined (Table 6). The highest texturization was obtained when butternut squash and parsnip puree was incubated with the fungal pectin methylesterase and papain. In contrast, butternut squash and parsnip puree incubated with Bromelain led to a small texture increase compared to the purees incubated without any enzyme preparation.

Table 6: Experimental setup to assess the impact of fungal or plant pectin methylesterase- containing enzyme preparation on the texturization of a butternut squash and parsnip puree.

Examples 100 - 123

Method of making a texturized butternut squash puree and parsnip puree incubated with three different fungal and a plant pectin methylesterase-containing enzyme preparation

(Papain)

The effect of three different fungal (derived from Aspergillus species) and a plant pectin methylesterase-containing enzyme preparation (Papain) on the texturization of butternut squash and parsnip puree in the presence of calcium chloride (0.4 %(w/w)) was determined using a texture analyzer (Penetration force (N) determined between 15 - 20 mm strain) (Table 7). In general, the incubation of both purees with papain led to a 2 to 3-fold higher required force to penetrate the puree with a probe compared to the incubation of these purees with fungal-derived pectin methylesterases. No significant difference in the penetration force was determined among purees incubated the different fungal-derived pectin methylesterases.

Table 7: Experimental setup to assess the impact of three different fungal and a plant pectin methylesterase-containing enzyme preparation (Papain) on the texturization of butternut squash and parsnip puree

Examples 124 - 130

Method of making a texturized apple puree incubated with 6 different commercial plant- derived pectin methylesterase-containing enzyme preparation from papaya (Papain)

The effect of 6 different commercial plant-derived pectin methylesterase-containing enzyme preparation from papaya on the texturization of apple puree in the presence of calcium chloride (0.4 %(w/w)) was visually determined (Table 8). All papain preparations led to a texturization of commercial apple puree in the presence of calcium chloride.

Table 8: Experimental setup to assess the impact of 6 different commercial plant-derived pectin methylesterase-containing enzyme preparation from papaya (Papain) on the texturization of commercial apple puree. Examples 131 - 136

Method of making texturized fruit and vegetable puree-based shapes - starting from fresh puree using papain

Examples were prepared using freshly prepared fruit and vegetable purees. The texturization was achieved by incubation of the fruit and vegetable purees in the presence of calcium using papain. The process is described under Example 1. A summary of the prepared prototypes is presented in Table 9.

Table 9: Shaped texturized purees using various self-made fruit and vegetable purees

Examples 137 - 139

Method of making texturized fruit and vegetable puree-based shapes starting from commercial puree

Examples were prepared using commercial fruit and vegetable purees. The texturization was achieved by incubation of the fruit and vegetable purees in the presence of calcium using papain. The process is described under Example 2. A summary of the prepared prototypes is presented in Table 10. Furthermore, the prepared prototypes were freeze-dried. Moreover, the nutritional values have been calculated for the shaped and freeze-dried prototype, which are presented in Table 11.

Table 10: Shaped texturized purees using various commercial fruit and vegetable purees

Table 11: Nutritional composition of a freeze-dried texturized purees (bear shape) using a commercial fruit puree (Table 10)

Example 140 - 145

Method of making texturized fruit and vegetable puree-based snacks - starting from commercial puree which were enriched in grains or yoghurt using additional freeze-drying using papain

Commercial fruit and vegetable purees that were enriched with rice flour (Example 140 & 141) and hydrolyzed wheat flour (~56%) with milk powder addition(Example 142 & 143), which led to a final cereal concentration of 25% in the product after freeze drying, could be texturized after the incubation with papain in the presence of calcium. The incubation of these ingredients without papain addition did not lead to a texturized mass. If 50% yoghurt was added to the fruit puree, the mass could be texturized after the incubation with papain in the presence with calcium (Example 144). In contrast, the incubation without papain of these ingredients did not lead to a texturized product. The addition of a higher yoghurt amount (i.e. 90%) to fruit purees hindered the texturization using papain in the presence of calcium (Example 145) (Table 12).

Table 12: Fruit and vegetable puree-based snacks with rice flour, hydrolyzed wheat flour (~56%) with milk powder addition or yoghurt

Examples 146 - 157

Method of making texturized fruit and vegetable puree-based snacks - starting from commercial puree which were enriched in grains using additional freeze drying and papain

In addition to Example 140 - 145, commercial fruit and vegetable purees were enriched with either oat flour (Examples 146 - 151) or hydrolyzed wheat flour (~56%) with milk powder addition (Examples 152 - 157). The objective was to determine if the addition of papain had a positive effect on the freeze-dried product texture compared to the samples that were incubated without papain addition. The cereal concentration of the final products ranged from 25 - 50% (Table 13).

All 12 samples were freeze dried after incubation with and without papain. The results of the visual inspection and sensory test are summarized in the table below. Table 13: Healthy Snacks (bear shape) with oat flour & hydrolyzed wheat flour (~56%) with milk powder addition

Examples 158 - 159

Method of making plant-based vegetable rolls using parsnip puree, calcium, and papain as a binder

The objective of this research was to create vegetable rolls using a vegan based binder. Here, the approach was to use papain to create a binder that was based on parsnip puree which was enriched with citrus fiber and calcium chloride to texturize vegetable rolls. The process to texturize vegetable rolls and burger patties using the binding technology that was based on parsnip puree, citrus fiber, CaCl2 and papain is described in Figure 3. Parsnip puree was prepared as described for Example 1.

Two sets of prototypes were prepared for each concept (Examples 158a & 158b). The first set of samples followed the process as described in Figure 3. The second set of prototypes were prepared without any enzyme incubation step, as this reduced the process time for one hour. In the latter case, prototypes were immediately heat-treated to deactivate the papain preparation. Compared to Example 158, the strongest texturization was achieved for Example 159, in which the used binder consisted of parsnip puree, citrus fiber, calcium chloride and papain (Table 14, 15 & 16).

Table 14: Ingredient list and binder mass to prepare vegetable rolls

Table 15 Cutting force (g) of the vegetable rolls after 24 h (frying, no freezing, measured at 20°C)

Table 16: Cutting force (g) of the vegetable rolls after 24 h (after freezing, thawing, and frying, measured at 60°C)

Examples 160 - 162

Method of making plant-based patties with chickpeas and rice using parsnip puree, calcium, and papain as a binder The aim was to texturize these burger patties based on the developed binder using the papain-based enzyme technology. Here, the approach was to use papain to create a binder that was based on parsnip puree enriched with citrus fiber and calcium chloride. The process is described in Figure 3. Parsnip puree was prepared as described for Example 1. Three examples were prepared. The main difference between these examples is the used binder. Example 160 contains a binder that is based on parsnip puree, calcium chloride, citrus fiber and papain. Compared to sample 160, parsnip puree was replaced by water for Example 161.

Example 162 is based on the binder of Example 160 without the addition of papain. Based on the cutting force that is required to completely cut half of a burger into two pieces, the patty of Example 160 is the most texturized prototype. In particular, the storage of Example 160 at 4°C after baking led to the most texturized patty. Table 17: Ingredient list of the burger patty examples

Table 18: Required cutting force (g) of the burger patty examples

Examples 163 - 164

Method of making plant-based patties vegetables using parsnip puree, calcium, and papain as a binder

Based on the results of Examples 160 - 162, burger patties with rice and ratatouille were prepared. The process is described in Figure 3. Parsnip puree was prepared as described for Example 1. The use of parsnip puree, citrus fibre, calcium chloride and papain as a binder lead to a good texturization (Example 163). The created burger patties of Example 163 were stable upon freezing and thawing, as well after frying. The preparation of the Example 164 without papain addition led to a soft burger that did not pass the fork test after frying.

Table 19: Ingredient list of the burger patty examples with rice and ratatouille

Examples 165 - 168

Method of making texturized fruit and vegetable puree-based fillings for confectionery applications using papain

The objective of this approach was to develop a filling that consists of texturized fruit and vegetable purees which were enriched with calcium and fibers using papain for confectionery applications, like chocolate fillings. In a next step, this texturized mass was covered with milk or dark chocolate.

Table 20: Summary of the prepared experiments, including recipes and sensory evaluation.

Example 169 Method of thickening fruit and vegetable purees using papain

To determine the thickening effect of the papain preparation on fruit and vegetable purees, a bench scale setup using small volumes was developed. Therefore, a drilling machine with a propeller for mixing of the samples and a water bath were used. In general, 20 to 25 g of fruit and vegetable puree was weighed in and cooled (4 - 6°C). After cooling, the papain preparation, which was either powder or liquid, was added to the puree. After rigorous mixing of the samples for 30 seconds, using a drilling machine and a tailor-made stirring rod, samples were incubated in a water bath. The enzyme reaction was stopped by placing the incubates into a water bath that was pre-set to a temperature of 90°C for at least 10 min. The texture of the samples was evaluated visually, by placing the samples on a plastic rack, or, by using a texture analyzer. For the bench scale experiments, a positive control was used as a reference (texturized puree). This sample was obtained by adding calcium chloride (0.1 M; for CaCl2 x 2 H2O, 277 mg per 25 g of puree) to the puree and using 0.4% of a papain preparation. Larger samples (125 to 150 g) were prepared using glass jars. A larger amount allowed the use of a Bostwick Viscosimeter Consistometer. An overview of the general process flow for the thickening of fruit and vegetable purees using a papain is shown in Figure 4.

Examples 170 - 193

Method of thickening apple purees using papain and salts at varying pH values

The effect of pH and salt addition on the papain-mediated apple puree thickening was assessed by adding 0.1 M of calcium chloride or sodium chloride to apple puree at varying pH values, ranging from 3.2 to 6.1 (Table 21). In the presence of calcium chloride, the addition of papain led to the strongest puree thickening compared to purees incubated with or without salt addition and papain. The higher the pH value, the stronger was the effect on the papain- mediated puree thickening for all incubation conditions that were tested (with or without salt addition). Sodium chloride addition led to an increased papain-mediated puree thickening at a pH value between 4.7 to 6.1. The observed effect of the varying parameters on the on the papain-mediated puree thickening are summarized in Table 21, using a subjective classification system ranging from '+++' (strongest texturization) to (no texturization).

Table 21: Experimental setup to determine thickening apple purees using papain and salts at varying pH values

Examples 194 - 213

Method of thickening apple purees using different papain concentrations and pH values

The effect of varying papain concentrations (0.1 - 4.0%, (w/w)) on the apple puree thickening was determined at pH 3.5 and 6.0 (Table 22). At pH 6.0, the addition of 0.4 to 4.0% (w/w) papain led to a thickening of apple puree, whereas a concentration of 1.0% papain was needed to enhance the viscosity of the apple puree at pH 3.5.

Table 22: Experimental setup to determine the effect of varying papain concentrations (0.1 - 4.0%, (w/w)) at pH 3.5 and 6.0 on the thickening of apple puree.

Examples 214 - 220

Method of thickening apple purees using different papain concentrations and the Bostwick Consistometer

The effect of varying papain concentrations (0.0 - 1.4%, (w/w)) on the apple puree thickening was determined at the natural pH of about 3.8 (Table 23, Figure 5). Therefore, 120 g of apple puree was incubated with a papain preparation using 500 mL glass flasks without agitation. After the incubation, the puree samples were heat-treated in a water bath under agitation until the puree reached a temperature of 90°C, followed by a holding time of 10 min. The samples were cooled down at room temperature overnight. Based on the Bostwick analysis, the addition of 0.6 to 1.4% (w/w) of papain to the apple puree led to an increase in the viscosity of the puree after incubation. The thickest purees were formed when apple puree was incubated with 1.0% or 1.2% papain. To determine the effect of shearing on the papain- mediated apple puree thickening, the apple puree samples that was thickened with 1.2% papain was stirred for 30 seconds using a drilling machine device with a propeller (~4 cm diameter). The stirring of the puree diminished the papain-mediated viscosity increase of the apple puree (dashed line, Figure 5). Table 23: Experimental setup to determine the effect of varying papain concentrations (0.0, 0.4, 1.0 and 2.0%, (w/w)) on the apple puree thickening.

Figure 5 shows Bostwick analysis of apple puree incubated with various papain concentrations for 60 min.

Examples 221 - 229

Method of thickening a commercial banana puree using papain at varying pH values

The effect of pH on the papain-mediated thickening of banana puree was investigated by the incubation of the puree with and without papain and calcium chloride at 45°C for 60 minutes (Table 24). The incubation of banana puree with papain and calcium led to a strong texturization of the puree at all three pH values which were tested. In comparison, the incubation of banana puree with papain only led to a viscosity increase which was pH dependent and the highest viscosity increase was achieved at pH 6.20. The pH did not influence the viscosity of the banana puree in the absence of papain and calcium.

Table 24: Experimental setup to determine the effect of pH on the papain-mediated thickening of banana puree in the absence or presence of papain and calcium chloride.

Examples 230 - 250

Method of thickening self-made carrot purees using different papain dosages

Fresh carrot purees were prepared based on orange and black carrot using the Thermomix as described in Example 1. In total, four different purees were obtained (Table 25). As carrots have a pH above 5.0, one version of each puree was prepared in the presence of lemon juice to decrease the pH below 5.0 (~pH 4.8). Prior to the DoE, the pH of all carrot purees was adjusted to 5.0 using lemon juice.

Table 25: Experimental setup to prepare carrot purees from fresh carrots.

Overall, the preparation of carrot purees from fresh carrots led to a thicker puree compared to commercial puree products. The main reason for this difference in thickness is that a less severe heat-treatment was applied using the Thermomix compared to an industrial process. The incubation of all four purees with papain at a concentration of 0.4 - 1% (w/w) papain or above led to strong thickening to the purees (45°C, 60 minutes incubation time) (Table 26).

Table 26: Experimental setup to prepare carrot purees from fresh carrots.

Example 230

Summary of the crude pectin methylesterase-containing plant preparations and their protein content

The protein content of the enzyme preparations was determined by using the BCA Protein Assay Kit (Pierce™ BCA Protein Assay Kit, Thermo Fisher Scientific, Rockford, IL, USA) and bovine serum albumin (BSA) was used for calibration. In addition to the BCA analysis, the protein content was also calculated based on the determined nitrogen content using Dumas (FlashSmart Elemental Analyzer, Thermo Fisher Scientific, USA) and the nitrogen conversion factor 6.25. The determined protein content of 10 pectin methylesterase-containing plant preparations ranged from 1.0-93.0% w/w based on DUMAS and the BCA assay. The powdered preparations (papain) that derived from papaya showed the highest protein content. The relative standard error of other protein determinations was lower than 5%. Table 27: Overview of the protein content of commercial pectin methylesterase-containing plant preparations