The present invention relates to the field of biodegradable, ecologically-friendly consumer goods. In particular, the invention provides a pasta structure comprising at least one enzyme capable of stabilizing the pasta structure, wherein the enzyme is selected from the group comprising a carboxylester hydrolase, a transglutaminase, an oxidase or an oxygenase. The enzyme may reduce or prevent the loss of starch from the pasta when the structure is contacted with water. The pasta structure is suitable for use as a food handling device and may e.g. have the shape of a straw, a bowl, a cup, a plate, a spoon, a fork, a knife, a jar, a funnel, a swizzle stick or a cone. It typically comprises water and wheat selected from the group comprising Triticum aestivum, Triticum durum, Triticum dicoccum, Triticum spelta, Triticum monococcum and a mixture thereof. The invention further provides a method of preparing said pasta structure and a use of at least one enzyme capable of stabilizing a pasta structure, wherein the at least one enzyme is selected from the group comprising a carboxylester hydrolase, a transglutaminase, an oxidase or an oxygenase, for stabilizing the pasta structure. Finally, use of the pasta structure as a food handling device is disclosed, preferably as a drinking straw.

The pollution of our environment with plastic waste constitutes one of the biggest challenges of our time. According to numbers released by the European Parliament, the yearly world-wide plastic production has increased from 1.5 million tonnes in 1950 to 322 million tonnes in 2015. (www.europarl.europa.eu/news/en/headlines/soci- ety/20181212STO21610/plastic-waste-and-recycling-in-the-eu-f acts-and-figures). The total amount of plastic produced in this 65-year period is estimated at 8.3 billion tonnes. 22 % of the prepared plastic is used for consumer and household items. A study conducted by the Heinrich-Boll-Stiftung in cooperation with the Bund fur Umwelt und Naturschutz Deutschland (BUND) concluded that in Germany alone, each person produces 38 kg plastic waste per year (www.bund.net/fileadmin/user_upload_bund/publikationen/chemi e/chemie_plasti- katlas_2019.pdf). Less than a third of the plastic waste in Europe is recycled. However, plastic recycling itself faces several challenges. For instance, the diversity of plastics that are often customized to the particular needs of manufacturers complicates the recycling process, making it costly and affecting the quality of the end product (www.europarl.eu- ropa.eu/news/en/headlines/society/20181212STO21610/plastic-w aste-and-recycling-in-the- eu-facts-and-figures). Therefore, the vast majority of the world-wide plastic waste is disposed of after single use. However, plastic takes hundreds of years to biodegrade and the resulting microplastics contribute to environmental damage, especially in the marine ecosystem, as approximately 4-12 million tonnes of plastic waste enter the oceans every year. Recently, scientists from the University of Manchester analyzed the amount of microplastic in the Thyrenian Sea and detected microplastics in every seafloor sample taken, with microplastic concentrations reaching up to 1.9 million pieces per square meter (Kane et al., 2020. Seafloor microplastic hotspots controlled by deep-sea circulation. Science 368(6495), 1140- 1145).

These and other alarming figures have, however, initiated a slow rethinking among many consumers and companies. Consequently, environmentally friendly alternatives to products made of plastic are in great demand. Some governments have already banned the use of e.g. plastic drinking straws. The European Union decided to ban cutlery, tableware, straws and cotton buds made from plastic as well as food packaging made of polystyrene starting from July, 2021. Even large fast-food chains have recognized this development and started to prohibit the use of plastic straws in many of their restaurants.

A large number of environmentally friendly and oftentimes reusable alternatives have been developed for many of the above mentioned plastic objects, including biodegradable cutlery made of wood, bamboo or bioplastic. Drinking straws may be made of paper, glass, stainless steel, bamboo or even actual straw. Wisefood offers edible straws, spoons and swizzle sticks made of grain, apple fibers and stevia (www.wisefood.eu). However, many of these ecological alternatives are associated with drawbacks. Straws made of paper are becoming increasingly popular, but lose their stability very quickly when used. Reusable items made of metal, glass or bamboo on the other hand are relatively expensive and make it necessary for the user to have the aid with him at all times and to wash it after each use. Alternatively, fast-food restaurants would have to provide these items and clean them after each customer, which would result in high costs and an increased energy consumption. Finally, many of the proposed alternatives to plastic often exhibit an unpleasant taste of their own and could thus be rejected by customers.

Childhood experience suggests the use of hollow noodles as drinking straws. This has been taken up, for example, by AU 2018101026, which describes a straight or bent straw of wheat or gluten-free material. The straw may come in different shapes and flavors and is intended to be completely edible.

WO 2020/069587 teaches the production of a drinking straw made of durum wheat. Pasta made from durum wheat (Triticum durum) is generally characterized by a higher resistance to cooking water compared to pasta made from Triticum aestivum.

Similar pasta straws made of durum wheat are commercially available from various websites e.g., makkaroni-strohhalme-kaufen.de, www.sausalitos.de/shop/sausaroni-pasta- strohhalme~p44976, www.vomfass.de/pasta-strohhalme, www.pastastraws.org, stroodles.co.uk or www.drinkstuff.com/products/product. asp?ID=27441 .

The pasta straws disclosed in AU 2018101026 and W02020/069587 or sold on the internet usually last for approximately one hour in a cold beverage kept at room temperature before losing their stability and starting to become soft. While a period of one hour may be considered long enough to finish a normal glass of water or a soft drink, the consumption of a large and calorie-rich cocktail or milkshake on convivial occasions can, however, easily exceed this period. In consequence, the straw made of pasta eventually starts to break and clog, which will significantly reduce the drinking pleasure for the consumer.

To improve its moisture resistance, W02020/044049 therefore suggested treating a pasta straw with an edible wax. The wax should be incorporated into the pasta dough. This requires quite high amounts of wax to be added, which leads to higher costs. In addition, the product no longer corresponds to classic pasta products in its composition.

Collectively, although a growing number of customers has developed an increased environmental awareness and thus wants to reduce their consumption of products made of plastic, many of the existing alternatives are either comparably expensive or lack the stability and durability of familiar plastic items. Also, even many so-called biodegradable items made of paper or biodegradable plastic may still take months or even years until they are fully degraded. Even though straws made of pasta are becoming more and more popular due to their neutral taste and the possibility to modify their appearance by use of e.g. food colorants, the low water-resistance of dry pasta limits the time period the straws may be used. For the same reason, dry pasta usually is not used for manufacturing other food handling devices such as cutlery or plates. Therefore, there is a demand for environmentally friendly and rapidly degradable food handling devices that can be cheaply produced while meeting the expectations of costumers with regard to stability and moisture-resistance. This problem is solved by the present invention, especially by the subject matter of the claims.

The present invention provides a pasta structure comprising at least one enzyme capable of stabilizing the pasta structure, wherein the enzyme is selected from the group comprising a carboxylester hydrolase, a transglutaminase, an oxidase or an oxygenase.

In the context of the invention, the term structure is intended to refer to a shaped or formed article or component thereof, i.e. an object or part thereof which has been formed, e.g., extruded, moulded, kneaded, pressed, pulled, rolled, blown, cut or punched out into a desired shape, preferably, pressed. The structure may thus also be, e.g., a moulded article. Preferably, the shaped pasta structure is a food-handling device.

The pasta structure may, e.g., be selected from the group comprising a straw, a bowl, a cup, a plate, a spoon, a fork, a knife, a jar, a cone, a funnel and a swizzle stick. Preferably, the pasta structure is a straw, wherein the straw is usable for drinking a beverage, i.e., a drinking straw. In its simplest form, the straw has a cylindrical shape and is hollow inside to allow the passage of a liquid, i.e., it is a rigid tube formed of pasta. The drinking straw may preferably have a length of 5-30 cm, e.g., of about 10 cm, about 15 cm or about 20 cm and a total diameter of 3-15 mm, preferably of 5-12 mm, most preferably of about 10 mm. It may be a straight straw. Alternatively, it may also comprise a bend to facilitate the drinking process.

The structure according to the invention, i.e., the food-handling device, is preferably made from pasta and consists of pasta. Alternatively, it may also comprise pasta in combination with at least one other component. Pasta is the Italian designation for a noodle rich in starch that is usually prepared from a dough comprising wheat semolina and water. In some recipes, the dough may further comprise eggs. Pasta may exist both in fresh (pasta fresca) and in dried form (pasta secca). The pasta structure of the invention typically is dry pasta. According to the Encyclopedia of Pasta released by the University of California Press, there are more than 300 officially listed shapes of pasta (Zanini de Vita, O., 2009. Encyclopedia of pasta, Volume 26, University of California Press). Therefore, pasta dough constitutes an excellent basic substance for forming or shaping a pasta structure into any of the objects or articles listed above.

The pasta structure of the invention typically comprises water and wheat selected from the group comprising Triticum aestivum, Triticum durum, Triticum dicoccum, Triticum spelta, Triticum monococcum and a mixture thereof. Alternatively, it may also comprise other cereal products, e.g., barley, rye, rice, corn, sorghum, teff, ricegrass, quinoa, chia, oats, millet or amaranth as well as products from other food plants such as beans, peas, lentils, buckwheat, linseed, sweet potato, potato, arrowroot, cassava or a combination thereof.

Triticum aestivum, better known as common wheat or bread wheat, constitutes approximately 90 % of the globally produced wheat. In the context of the invention, it is also referred to as soft wheat (in contrast to durum - hard - wheat). T. aestivum can also be used for products other than bread, cake, cookies, biscuits and crackers. It is a hexapioid wheat species and contains on average about 11 % protein (dry weight) (Zilic et al., 2011. Characterization of proteins from grain of different bread and durum wheat genotypes. Int. J. Mol. Sci. 12(9), 5878-5894). Gluten constitutes about 75-85 % of said total grain proteins and stores carbon, nitrogen and sulphur to support seed germination. Gluten is a protein mixture composed of prolamins and glutelines, which, in the context of wheat, are usually referred to as gliadins and glutenins, respectively. Glutenins form protein aggregates stabilized via intermolecular disulfide bonds that become attached to the monomeric gliadins. Collectively, gluten proteins form a matrix with viscoelastic and adhesive properties (Shewry et al., 2002. The structure and properties of gluten: an elastic protein from wheat grain. Phil. Trans. R. Soc. Lond. 357, 133-142). T. aestivum carries rather soft grains that can be conveniently milled into fine flour. As a result, the gluten networks become exposed and can be easily brought together during the mixing and kneading of the dough, thereby creating new protein-protein interactions within the growing gluten network. Accordingly, doughs prepared from T. aestivum are usually high in strength and elasticity and are therefore preferred for bread making.

Traditionally, the wheat species used for preparing pasta dough is Triticum durum. T. durum is a tetrapioid wheat species, probably derived from the tetrapioid species Triticum dicoc- cum, and the second most cultivated species of wheat after T. aestivum. The Latin name Durum translates as hard, as the grains of T. durum are known for being highly resistant to milling. T. durum is therefore mostly processed into more coarse-grained semolina rather than into fine flour. T. durum is described to contain slightly higher levels of gluten compared to T. aestivum (Zilic et al., 2011). However, because the gluten- and starch-rich endosperms are often only partly cracked in Durum semolina, the gluten is less readily available. Accordingly, dough made from T. durum is only rarely used for baking bread and is instead particularly suitable for making pasta, as its lower elasticity facilitates easier shaping and cutting. Triticum dicoccum, better known as emmer wheat or hulled wheat, is considered to be one of the oldest domesticated crop species. Emmer is a particular stress-resistant wheat and therefore can grow on comparably poor soils. Similar to T. durum, emmer possesses relatively hard grains. Emmer wheat is most famously employed in the production of a particular type of Italian bread (pane di farro), but may also be used for garnishing soups or for preparing beer.

Triticum spelta, also referred to as spelt or dinkel wheat, is another relict crop, which enjoys growing popularity among more health-oriented consumers. Spelt is a hexapioid wheat and is most commonly used for baking breads, rolls and other pastries. It may also be employed in beer brewing or for the distillation of spirits.

Similar to emmer and spelt, Triticum monococcum (einkorn wheat) is an ancient wheat and is characterized by a high protein and fat content. It is commonly consumed in Provence, France and may be used as an ingredient of bulgur.

Emmer, spelt or einkorn wheat are suitable for being processed into pasta. However, pasta products made from these wheat species merely represent niche products that are sold at a comparably high price. In the present invention, the preferred wheat species used for preparing the pasta structure preferably is Triticum aestivum. T. aestivum is by far the most cultivated wheat species in the world, and it is therefore considerably cheaper and far more available than T. durum, T. dicoccum, T. spelta and T. monococcum. Accordingly, using T. aestivum for producing the pasta structure thus reduces production costs and results in a final product that may be sold at a lower price. The inventors found that pasta structures can be advantageously prepared using a combination of T. aestivum and the enzyme as described herein for the preparation of pasta structures, without addition of other cereal components such as T. durum. In particularly, the pasta structures formed are very stable and can advantageously be used as food handling devices, e.g., as straws.

However, the inventors found out that stability of pasta structures made from T. durum is also improved by the combination with the enzyme. Thus, the pasta structure may comprise T. durum, e.g., it may comprise a mixture of T. aestivum and T. durum. Preferably, the pasta structure comprises more T. aestivum than T. durum. The percentage of T. aestivum in the pasta dough may be, e.g., 0-100 %, e.g., 90-100 %, 80-90 %, 70-80 %, 60-70 %, 50-60 %, with T. durum ad 100 %. % refers to a weight per weight ratio (wt/wt) unless otherwise mentioned. In other embodiments, the pasta structure may however also comprise more T. durum than T. aestivum, i.e. , the percentage of T. aestivum being 40-50 %, 30- 20 %, 20-30 %, 10-20 % or 0-10 %, with T. durum ad 100 %. It may also comprise only T. durum and no T. aestivum.

The pasta of the invention may also comprise one or more of T. dicoccum, T. spelta and/or T. monococcum. The pasta may therefore also have a T. dicoccum, T. spelta and/or T. monococcum content of, e.g., 0-100%, e.g., 90-100 %, 80-89 %, 70-80 %, 60-70 %, 50- 60 %, 40-50 %, 30-40 %, 20-30 %, 10-20 % or 0-10 %, with any other of the herein described wheat species described herein, e.g., T. aestivum, ad 100 %.

The wheat product used for preparing the pasta dough may be flour, semolina or a mixture thereof.

Flour refers to a fine powder obtainable by grinding or milling raw wheat grains to a particle size of preferably approx. 100 to 250 pm. Smaller particles may also be contained. Cereal flour may either be whole grain, i.e., it may be prepared from the endosperm, germ and bran together, or it may be a refined flour, i.e., it may be prepared only or partly from the starch-rich endosperm. Typically, for use of the pasta structure as a food-handling device, refined flour prepared from the endosperm is preferred, e.g., corresponding to Type 550 (DIN-Norm 10355, 1992), because it is cheaper than most other flour types due to its common use a bread flour.

Semolina refers to the coarse, purified wheat midlings usually obtainable from milling hard wheat species, e.g. T. durum. During semolina production, the bran and germ of the wheat are flaked-off, while the starch-rich endosperm is cracked into coarse fragments. These endosperm pieces form the actual semolina when separated from the bran. The semolina can optionally be further ground into finer particles to produce flour.

The average particle size of semolina and flour may vary considerably depending on the employed milling technique and the type of wheat used. However, the mean particle size of semolina, e.g., from T. durum, is commonly in the range of more than 250 pm to about 750 pm (en.wikipedia.org/wiki/Semolina; Sacchetti et al., 2011. Effect of semolina particle size on the cooking kinetics and quality of spaghetti. Proc. Food Sci. 1 , 1740-1745).

The mean particle size is preferably analyzed by sieving using, e.g., a test sieve shaker such as a Ro-Tap® (www.haverparticleanalysis.com/en/sieve-analysis/ro-tapr-tes t-sieve- shaker/), Vibratory Sieve Shaker AS 200 Control (www.retsch.com/products/sieving/sieve- shakers/as-200-control/function-features/) or an air jet sieve such as the Laboratory Air-jet Lab sieve KLS (gkm-net.de/en/laboratory-air-jet-lab-sieves.html). Alternatively, particle size may also be determined by laser diffraction or spectrometry (Hareland, G.A., 1994. Evaluation of flour particle size distribution by laser diffraction, sieve analysis and near-infrared reflectance spectroscopy. J. Cereal Sci. 20(2), 183-190).

In one embodiment, the wheat used for preparing the pasta dough is provided as flour with an average particle size of less than 250 pm, preferably less than 200 pm, less than 175 pm or less than 150 pm, more preferably less than 125 pm, less than 110 pm or less than 100 pm. The finer the particle size of the ground wheat, the larger the reaction surface for the enzyme present in the pasta structure, which is advantageous. E.g., at least 50 %, preferably at least 75 %, at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 96 %, at least 97 %, at least 98 % or at least 99 % of the wheat is provided as flour e.g., as defined herein. Optionally, 100 % of the wheat used for preparing the pasta dough is provided as flour. Accordingly, typically, less than 50 %, preferably, less than 25 %, less than 20 %, less than 15 %, less than 10 %, less than 5 %, less than 4 %, less than 3 %, less than 2 % or less than 1 % of the wheat are provided as semolina. Optionally, the wheat product used for preparing the pasta dough does not comprise any semolina at all.

In an alternative embodiment, the proportion of semolina is higher, e.g., at least 25 %. It may for instances constitute at least 30 %, at least 35 %, at least 40 %, at least 45 % or at least 50 % of the wheat used for preparing the pasta dough. In some embodiments, the majority of the wheat used for preparing the pasta dough may be provided as semolina, e.g. at least 60 %, at least 70 %, at least 80 %, at least 90 %, at least 95 %, at last 95 %, at least 96 %, at least 97 %, at least 98 % or at least 99 %. Optionally, the entire wheat used for preparing the pasta dough is provided as semolina. Preferably, the semolina used for the pasta dough of the invention has an average particle size not larger than 500 pm, more preferably, smaller than 450 pm, smaller than 400 pm, smaller than 350 pm, smaller than 300 pm, or smaller than 275 pm.

The pasta structure of the present invention further comprises an enzyme capable of stabilizing the pasta structure, preferably added to the flour or into the dough prior to forming the structure. Enzymes have been previously used to reduce stickiness of cooked pasta made from T. aestivum (www.prvhh.de/newsroom/muehlenchemie/de/pressemeldung/news/d e- tail/News/pastazym-pd-erzielt-starke-verbesserung-der-kochto leranz-von-pasta/).

However, in the context of the present invention, it has been found that the enzyme surprisingly also increases stability of the uncooked pasta structure, and thus prevents the uncooked pasta structure from losing its structural integrity, especially when brought into contact with a liquid, preferably cold water. The pasta structure of the invention thus is an uncooked or raw pasta structure.

The stability, including the mechanical strength of a pasta structure, may be defined by its ability to withstand an externally applied mechanical force. This may be directly tested, for example, with the help of a measuring knife or stamp made of e.g. plastic, aluminum or steel, which penetrates or compresses the pasta structure at a defined path length (cf. e.g. texturetechnologies. com/industries/food-texture-analysis/pasta#:~:text=XTPIus%20 Tex- ture%20Analyzer%20is%20accurate,will%20accept%20your%20test% 20results). The higher the stability or strength of the pasta structure, the more mechanical force is required to penetrate the pasta with a blade or press the pasta with the stamp through a die. Prolonged exposure to water continuously softens the dry pasta and thus reduces the force needed to penetrate or compress the pasta. The mechanical strength can be tested in the presence or absence of water, particularly, cold water having room temperature (i.e., 25 °C) or less. The inventors found that the enzymes as defined herein increase the stability and mechanical strength of the pasta structure exposed to water, e.g., as measured with the help of a measuring stamp or blade (or, preferably, both). More details of the analysis are described in the examples below.

Another characteristic associated with decreasing stability of pasta in the presence of water is the continuous loss of starch from the pasta matrix into the surrounding medium. Accordingly, the stability, including the structural integrity of the pasta structure, may also be tested by measuring the amount of starch that is washed out when the pasta is submerged in water (typically, cold water having room temperature, i.e., 25 °C or less). This can simply be done by observing the continuously increasing turbidity of the water comprising the pasta over a certain time period, e.g., 30 or 60 min. Alternatively and preferably, the increasing loss of starch from the pasta into the surrounding water can be demonstrated chemically, e.g. through the addition of Lugol's solution, as shown in the examples below. Lugol’s solu- tion reacts with the dissolved starch and leads to the formation of dark inclusion compounds, a reaction that is the stronger the more starch is released into the water. The inventors found that the enzymes as defined herein increase the stability and the integrity of the pasta structure, e.g., as measured based on detection of the starch loss from the pasta matrix. The inventors found that pasta comprising a carboxylester hydrolase, a transglutaminase, an oxidase and/or an oxygenase advantageously has such increased stability. The pasta structures of the invention retained comparably high resistance to mechanical stress after extended immersion in water, in particular water with a temperature lower than room temperature (i.e., 25 °C). Adding any of the above enzymes to the pasta dough also significantly decreased the loss of starch from the pasta structure in the presence of water. Therefore, the enzymes are able to stabilize the pasta structure according to at least one of the two assays described herein. In a particular preferred embodiment, the pasta structure comprising the at least enzyme should exhibit both prolonged mechanical resistance to an external force as well as reduced starch loss in the presence of water when compared to an equivalent pasta structure lacking said enzyme.

Thus, advantageously, the pasta structure is stabilized compared to a pasta structure not comprising the enzyme, when in contact with a water-containing liquid, in particular, a beverage selected from the group comprising water, milk, a fruit juice, a soft drink such as coke or lemonade, a milkshake, a smoothie, an ice tea, a non-alcoholic cocktail or an alcoholic beverage such as a cocktail. The enzyme of the invention also stabilizes the pasta structure in the presence of a food containing a liquid, e.g., water, such as a soup, yoghurt, ice cream, pudding, cottage cheese, skyr, milk rice, porridge, a sauce, cream or fresh cheese, wherein the food preferably is cold food.

Preferably, the liquid or food according to the invention is a cold liquid or cold food, e.g. a liquid or a food with a temperature not higher than 30 °C, preferably not higher than room temperature (about 25 °C). Preferably, the liquid or food according to the invention has a temperature below room temperature, e.g., between 0 and 20 °C. Temperatures in this range will prevent the denaturation of proteins that form the matrix of the pasta dough and limit the degree of starch gelatinization, i.e., the process of starch dissolution in water. However, the enzyme of the invention may additionally be able to stabilize the pasta structure in the presence of a hot liquid or a hot beverage, i.e., a liquid or beverage with a temperature higher than 30 °C, e.g., at least 35 °C, at least 40 °C, at least 45 °C, at least 50 °C, at least 55 °C, at least 60 °C, at least 65 °C, at least 70 °C or at least 75 °C. However, the stability of the pasta structure will decline more rapidly in the presence of hot liquids or foods than in the presence of cold liquids or foods.

Due to the activity of the enzyme, the pasta structure of the invention may remain in a cold liquid or food as defined herein for extended periods of time before becoming instable and thus unsuitable for use as a food handling device, e.g., a straw, according to at least one of the assays described herein. The pasta structure according to the invention may retain its stability in a liquid (e.g., water) for at least 1-3 h, e.g., at least 1.5 h, at least 2 h, or at least 2.5 h. In the presence of a food, the pasta structure may retain its stability for even longer time periods, e.g. for at least 2-4 h, e.g. at least 3 h, or at least 3.5 h.

Addition of the enzyme to the dough of the pasta structure according to the invention may increase the firmness or mechanical strength of pasta in the presence of water within the first 30 min by at least 5 %, preferably at least 10 %, at least 15 %, or at least 20 %. This can be tested by subsequently subjecting the pasta structure to mechanical stress as described above or in the example below.

The stability or strength of the pasta structure depends on the integrity of the pasta matrix. The term pasta matrix is intended to relate to the structural network, which is mainly formed by proteins such as gluten as well as the starch present in wheat. The pasta matrix is considered strong if, e.g., the structural protein network of the pasta is very dense, i.e. when it comprises multiple cross-links between individual proteins or protein aggregates.

A high stability of the pasta structure is further ensured if the starch present in the pasta matrix is protected from undergoing swelling and gelatinization in water. Starch is a polymer formed by the highly branched amylopectin and linear amylose. Both molecules are polysaccharides formed by large numbers of D-glucose units linked together via 1 ,4-alpha glyo- sidic bonds. The branched amylopectin furthermore comprises 1 ,6-alpha glycosidic bonds at every 24 ^th -30 ^th glucose unit. Starch molecules arrange themselves in semi-crystalline granules inside the plant. When preparing dough from wheat, starch acts not only as a filler in the continuous matrix of the dough, but also appears to form a bi-continuous network with proteins (Horstmann et al., 2017. Starch characteristics linked to gluten-free products.

Foods 6(4), 29-50). When dried pasta is brought into contact with e.g. cold water, it absorbs water and starts to swell. In addition, intermolecular bonds of starch molecules will break down, the starch dissolves and is progressively washed out of the pasta matrix. Loss of starch from the matrix reduces the stability of the pasta structure, and ultimately softens it. This process will be accelerated by high temperatures. The enzyme according to the present invention may be capable of reducing or preventing the loss of starch from the pasta when the structure is contacted with water, e.g., as described herein.

In the context of the invention, the phrase “reducing the loss of starch” means that the activity of the enzyme present in the pasta structure results in modifications of the pasta matrix that effectively limit the amount of starch washed out of the pasta structure. When contacted with e.g. water, the loss of starch from the pasta structure comprising the enzyme may be reduced by at least 15 %, at least 20 %, at least 25 %, at least 30 %, at least 35 %, at least 40 %, at least 45 % or, preferably, at least 50 % compared to the starch loss of an equivalent pasta structure lacking the enzyme. The reduction of starch loss as described herein may be even as much as 55 %, 60 %, 70 %, 80 % or 90 %.

In one embodiment, the inventors found an increase in stability of the pasta structure exposed to water when the pasta comprises at least one enzyme that is a carboxylester hydrolase.

Carboxylester hydrolases (also known as carboxylic ester hydrolases) are enzymes that catalyze the hydrolysis of carboxylic esters into alcohols and carboxylic acids. Representatives of this class of enzymes are lipases, which catalyze the hydrolysis of fats (lipids) into their basic components, e.g. triacylglycerol lipases (EC 3.1.1.1), phospholipases (EC 3.1 .1 .4, EC 3.1 .1 .32) or galactolipases (EC 3.1.1 .26).

Besides protein and starch, wheat comprises a variety of lipids. The majority of lipids in wheat are esters formed by glycerol and fatty acids. These so-called glycerol ipids may be triglycerides, mono- and di-galactosyldiglycerides, phospholipids such as A/-acylphosphati- dylethanolamine, phosphatidylethanolamine, phosphatidylglycerol, or phosphatidylcholine, diglycerides or mono acyl glycerides. Other lipids that can be found in wheat are free fatty acids as well as sterol-based lipids and glycosphingolipids (Morrison, W.R., 1994. Wheat lipids: structure and functionality. Bushuk W., Rasper V.F. (eds) Wheat. Springer). Without intending to be bound by the theory, upon addition to the pasta dough, triacylglycerol lipases may interact with the acylglycerides present in the dough and catalyze their hydrolysis into fatty acids as well as partial glycerides, i.e. , mono- and diglycerides. These partial glycerides act as emulsifiers, i.e., they possess a polar or hydrophilic part and a non-polar or hydrophobic part. Phospholipases and galactolipases likewise split off fatty acids, which are highly hydrophobic, from the corresponding polar lipids, hence increasing the polarity of the remaining phospholipid or galactolipid. The resulting lyso-lipids and partial glycerides may subsequently interact with the starch within the pasta dough and are capable of delaying its gelatinization and therefore the loss of starch from the pasta matrix in the presence of e.g. water. Furthermore, fatty acids also tend to react with the hydrophobic regions of helical starch molecules, altering their pasting properties (Kibar et al., 2014. Effects of fatty acid addition on the physicochemical properties of com starch. Int. J. Food Prop. 17(1), 204-218). In addition, partial glycerides may also interact with gluten to promote aggregation and crosslinking of the protein matrix, which further prevents the release of starch from the pasta into the surrounding liquid. A denser protein matrix may also reduce the uptake of e.g. water by the starch and thus suppress its swelling.

Therefore, the carboxylester hydrolase of the present invention preferably is a lipase capable of hydrolyzing lipids into fatty acids and mono- or diglycerides, or a phospholipase capable of hydrolyzing diacyl-phospholipids into fatty acids and monoacyl-phospholipids, or a galactolipase capable of hydrolyzing diacyl monogalactosides or diacyl digalactosides into the corresponding monoacyl galactosides.

Preferably, the enzyme of the invention comprises an amino acid sequence having SEQ ID NO: 1. Such an enzyme is commercially available as “Pastazym Duo Pure” from Muh- lenchemie GmbH & Co. KG. The enzyme may also comprise an amino acid sequence having at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, or at least 99 % sequence identity to SEQ ID NO: 1 , wherein the enzyme is capable of hydrolyzing lipids in pasta dough, e.g., as defined herein. The enzyme may also consist of SEQ I D NO: 1 .

Alternatively, the enzyme of the invention comprises an amino acid sequence having SEQ ID NO: 2. Such an enzyme is commercially available as "Pastazym Superflex" from Muh- lenchemie GmbH & Co. KG. The enzyme may also comprise an amino acid sequence having at least 80 % , at least 85 %, at least 90 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, or at least 99 % sequence identity to SEQ ID NO: 2, wherein the enzyme is also capable of hydrolyzing lipids in pasta dough, e.g., as defined herein. The enzyme may also consist of SEQ ID NO: 2.

In an alternative embodiment, the enzyme of the invention may be an oxidase selected from the group comprising a glucose oxidase and a sulfhydryl oxidase. These enzymes have also been found to stabilize the pasta structure of the invention. The name oxidase refers to enzymes capable of catalyzing oxidation-reduction reactions. The enzyme glucose oxidase was found to be capable of strengthening the gluten network in dough prepared from wheat. Glucose oxidase can catalyze the oxidation of D-glucose into gluconic acid and H2O2. The H2O2 in turn oxidizes thiol groups, in particular of glutenin proteins, to create new disulphide crosslinks. To catalyze these reactions, glucose oxidases require O2, which is both naturally present in wheat flour but is also introduced into the dough during mixing (Meerts et al., 2017. Enhancing the rheological performance of wheat flour dough with glucose oxidase, transglutaminase or supplementary gluten. Food Bioproc. Technol. 10(12), 2188-2198).

Accordingly, the enzyme of the invention may comprise an amino acid sequence having SEQ ID NO: 3 or SEQ ID NO: 4. An exemplary suitable enzyme of SEQ ID NO: 3 is commercially available as “Sternzym Gloxy”, and of SEQ ID NO: 4 as “Sternzym Gloxy TGO”, both from SternEnzym GmbH & Co. KG. The enzyme may also comprise an amino acid sequence having at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, or at least 99 % sequence identity to either SEQ ID NO: 3 or SEQ ID NO: 4, wherein the enzyme is also capable of strengthening the protein matrix of pasta dough, in particular via catalyzing the formation of disulphide crosslinks, e.g., as defined herein. The enzyme may also consist of SEQ ID NO: 3 or 4.

Similar to glucose oxidase, sulfhydryl oxidase uses molecular oxygen as electron acceptor to oxidize free thiol groups in proteins, promoting the formation of disulfide bonds.

Therefore, the enzyme of the invention may also comprise an amino acid sequence having SEQ ID NO: 5. Such an enzyme is commercially available as "Thiolase" from SternEnzym GmbH & Co. KG. The enzyme may also comprise an amino acid sequence having at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, or at least 99 % sequence identity to SEQ ID NO: 5, wherein the enzyme is also capable of strengthening the protein matrix of pasta dough, in particular via catalyzing the formation of disulphide crosslinks, e.g., as defined herein. The enzyme may also consist of SEQ ID NO: 5.

Transglutaminases have also been found to stabilize pasta structures of the invention. Thus, the enzyme of the invention may also be a transglutaminase.

The enzyme transglutaminase catalyzes the acyl-transfer reaction between £-amino groups of peptide-bound lysine residues and the y-carboxyamide group of peptide-bound glutamine residues. In consequence individual gluten chains may become permanently cross-linked via iso-peptide bonds (Meerts et al., 2017).

Therefore, the enzyme of the invention may also comprise an amino acid sequence having SEQ ID NO: 6. Such an enzyme is commercially available as “Sternzym PT 8001” from SternEnzym GmbH & Co. KG. The enzyme may also comprise an amino acid sequence having at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, or at least 99 % sequence identity to SEQ ID NO: 6, wherein the enzyme is also capable of strengthening the protein matrix of the pasta dough, in particular via catalyzing the formation of iso-peptide crosslinks, e.g., as defined herein. The enzyme may also consist of SEQ ID NO: 6.

Oxygenases have also been found to stabilize pasta structures of the invention. Thus, the least one enzyme of the invention is an oxygenase, e.g., a laccase.

Oxygenases oxidize their substrates by transferring oxygen atoms to them. Laccases are capable of oxidizing a large variety of aromatic compounds. The reaction products of laccases often continue to react non-enzymatically; accordingly, laccase favors the generation of polymers. Without intending to be bound by the theory, when added to dough containing wheat, laccase presumably catalyzes the formation of crosslinks between non-starch polysaccharides, such as ferulic acid-substituted arabinoxylan hemicellulose, which results in arabinoxylan network formation and thus improved dough resistance. In addition, laccase may oxidize the tyrosyl residues of gluten proteins or enhance the disulphide bridge formation in gluten polymers via ferulic acid-derived radicals. In consequence, protein aggregation in the dough is increased (Selinheimo, E., 2008. Tyrosinase and laccase as novel crosslinking tools for food biopolymers, PhD Thesis, VTT publications, 693).

Therefore, the enzyme of the invention may also comprise an amino acid sequence having SEQ ID NO: 7. Such an enzyme is commercially available as “Suberase” from Novozymes A/S. The enzyme may also comprise an amino acid sequencing having at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, or at least 99 % sequence identity to SEQ ID NO: 6, wherein the enzyme is also capable of strengthening the protein matrix of pasta dough and catalyzing the formation of an arabinoxylan network, e.g., as defined herein. The enzyme may also consist of SEQ ID NO: 7.

The enzyme of the invention may also be an enzyme other than a carboxylester hydrolase, oxidase, transglutaminase or oxygenase, as long as it is capable of reducing or preventing the loss of starch from the pasta when the pasta structure is contacted with water. However, preferably, said enzyme of the invention is a lipase. In another embodiment, the pasta structure may comprise a combination of any of the herein described enzymes. For instance, the pasta structure may comprise a lipase having at least 80 % sequence identity to SEQ ID NO: 1 and a glucose oxidase having at least 80 % sequence identity to SEQ ID NO: 3. The pasta structure may also comprise a combination of more than 2 different enzymes, e.g., at least 3, at least 4, or at least 5 different enzymes.

The enzyme of the invention may be present in the final dried pasta structure at a concentration of 1-500 mg/kg, preferably of 10-400 mg/kg, and more preferably 50-300 mg/kg. In a particularly preferred embodiment, the concentration of the enzyme in the dried pasta structure is about 100 mg/kg. The term “about” is intended to mean +/- 10 % in the context of the present invention.

The chemical reaction catalyzed by the enzyme of the invention requires the presence of water to occur. Therefore, prior to drying, the freshly prepared pasta dough comprising the enzyme preferably has an initial water content of 20-40 % (wt/wt), preferably 25-35 %. Most preferably, the water content of the pasta dough prior to drying is 30-33 %, e.g., about 31 %, as described in the example below.

After forming the freshly prepared pasta dough into the desired, herein defined shape, the resulting pasta structure is dried so that it is suitable for the use as, e.g., a food handling device according to the invention. Therefore, typically, the pasta of the final pasta structure is dry (i.e. , dried) pasta, i.e. it has a water content of 12.5 % or less.

The final water content of the dry pasta structure should not be higher than 14 %, because otherwise, the pasta structure would lack the required stability and would be prone to deformation or tearing. In addition, a water content higher than 14 % in the final pasta structure may increase the risk of mold growth. On the other hand, if the water content of the dry pasta structure were too low, the structure would be too brittle for use as a food handling device.

Accordingly, the dry pasta structure preferably has a water content of 5-14 %, e.g., of IQ- 13 % or about 12 % as described in the example below. The dried pasta structure may be stored at room temperature, i.e. at about 23 °C for at least 6 months, at least 1 year, at least 1 .5 years, at least 2 years, at least 2.5 years or at least 3 years before substantially changing its characteristics.

The invention further provides a method of preparing the pasta structure of the present invention, comprising steps of a) mixing ingredients for the pasta dough comprising water, wheat and the enzyme, b) forming the pasta dough into the structure, and c) drying the pasta structure until it has a water content of less than 14 % (wt/wt), based on the total weight of the final structure, and, d) optionally, packaging the pasta structure.

In step a), the wheat is preferably T. aestivum and provided as flour. As described above, the wheat may alternatively also be T. durum, T. dicoccum, T. spelta or T. monococcum. It may also be provided as semolina, or a mixture of semolina and flour. In an optional embodiment, the pasta dough may also comprise eggs. However, since the pasta structure of the present invention should be suitable for long-term storage, the pasta dough preferably does not comprise eggs. The dough may further comprise an edible color additive, e.g. a food color such as betanin, anthocyanin, beta-carotene, riboflavin, tartrazine, curcumin, indigo carmine, Allura Red AC, sepia, xanthin, xeaxanthin, lutein or Quinoline Yellow WS, or an extract from a plant, such as a spinach extract, a red beet extract, a carrot extract, or Calendula officinalis extract. Additional optional ingredients may be, e.g., a flavoring, e.g., sugar or sweetener, and/or a hydrophobic component such as a wax, e.g., beeswax. The enzyme of the invention is preferably added together with the other ingredients prior to mixing. Alternatively, it may be first added either to the water or the wheat product and premixed, before adding the wheat product or the water, respectively, and forming the dough. The enzyme may also be added after the water and the wheat have been mixed to create a dough. A possible recipe for the pasta dough is described in the example below.

As previously described, step b) may involve forming, extruding, moulding, kneading, pressing, pulling, rolling, blowing, cutting or punching the pasta dough into a structure, e.g., a food handling device as defined herein. A combination of any of these forming methods can also be applied. Pressing is a preferred embodiment of step b).

In step c), the pasta structure is dried to obtain a final structure with a water content of less than 14 % (wt/wt). Drying may take place at temperatures of up to 45 °C, up to 50 °C, up to 55 °C, up to 60 °C, up to 65 °C, up to 70 °C, up to 75 °C, up to 80 °C, up to 85 °C, , up to 90 °C, up to 95 °C, up to 100 °C, up to 105 °C or up to 110 °C, typically, until the desired water content of the final pasta structure is reached. Preferably, the pasta structure is dried at a temperature of up to 86 °C as described in the example below. To avoid the development of cracks in the pasta, drying should not be performed too fast. On the other hand, too slow drying may favor the growth of mold. Preferably the pasta structure should be dried for about 200-400 min, preferably for 250-350 min, more preferably for 275-325 min, e.g., about 300 min. Drying can be performed by any suitable method known in the state of the art, i.e. the pasta structure may first be pre-dried at room temperature before most of the moisture is removed in a final drying step. The final drying step may first comprise a step wherein the pasta structure is exposed to high temperatures and humidity, before in the second step, the temperature is slowly reduced and cold air is provided to stabilize the pasta structure. A possible drying process is presented in Tab. 2 and described in the example below.

After drying, the pasta structure according to the invention may optionally be impregnated with an edible layer selected from the group comprising a hydrophobic layer such as a wax, a coloring layer and a flavoring layer. The wax may be selected from the group comprising bayberry wax, candelilla wax, carnauba wax, castor wax, ouricury wax, rice bran wax, soy wax, tallow wax, beeswax or a combination thereof. The wax may further increase the durability of the pasta structure and prolong the amount of time the pasta structure can be contacted with a liquid or a liquid-containing food without significantly loosing stability. In addition, the hydrophobic layer, e.g., the wax, may contribute to a more familiar, smoother mouthfeel of the pasta structure (e.g., a straw, a spoon or a fork) that resembles that of an equivalent structure made from plastics. The coloring layer may comprise a food color additive, e.g., as defined above. The pasta structure of the invention is preferably taste-less, i.e., it will not affect or alter the taste of a beverage or food that is to be consumed with the help of the structure. However, if desired, the pasta structure may optionally be modified with a flavoring layer. The flavoring layer may be sweet, e.g., due to the addition of sugar. Alternatively, the flavoring layer may comprise a spice or a combination of multiple spices. The flavoring layer may e.g., be a wax as defined above that has been supplemented with, e.g., sugar or at least one spice and/or a coloring.

In an optional step d) the pasta structure may be packaged to facilitate transport and distribution of the pasta structure. To ensure maximum shelf-life, the pasta structure may be packaged in a vacuum-sealed package, e.g. a bag or a blister package. Optionally, the pasta structure is packaged in ecologically friendly, biodegradable package, e.g. a bag made of paper, or a bag of bio-degradable plastic.

The invention further teaches the use of at least one enzyme capable of stabilizing a pasta structure, wherein the enzyme is selected from the group comprising a carboxylester hydrolase, a transglutaminase, an oxidase or an oxygenase, for stabilizing a pasta structure. In particular, it is used for stabilizing an uncooked pasta structure. Said pasta structure can advantageously be used as a food-handling device, e.g., as a straw. The pasta structure of the invention is obtained, e.g., by preparing the pasta structure by the method as described herein.

Finally, the invention also provides a use of the pasta structure according to the invention or obtainable by the herein described method as a food-handling device. The food-handling device facilitates handling of a food or beverage, e.g. its consumption, carrying, stirring, storage or transport. Preferably, the food or beverage is cold, i.e. it has a temperature below 30 °C, most preferably below room temperature (25 °C). Most preferably, the pasta structure may be used as a drinking straw as defined herein.

In summary, the invention teaches a pasta structure, e.g., a food-handling device, comprising an enzyme capable of stabilizing the pasta structure to delay or prevent its destabilization in the presence of water or a water-containing liquid and/or food. As disclosed herein, enzymes such as carboxylester hydrolases, transglutaminases, oxidases or oxygenases are capable of modifying the pasta matrix in a way that reduces or even prevents the loss of starch from the pasta in the presence of water. The inventors were therefore able to provide an inexpensive, ecologically friendly and fully biodegradable alternative to food handling devices made of plastic, while avoiding many of the undesired drawbacks associated with other plastic-free alternatives known from the state of the art, in particular lack of stability and unpleasant off-tastes.

Throughout the invention, the term “about” is intended to be understood as ”+/- 10 %”. If “about” relates to a range, it refers to both lower and upper limit of the range. “A” is intended to mean “one or more”, if not explicitly mentioned otherwise.

All literature cited herein is herewith fully incorporated. The present invention is further illustrated, but not limited, by the following example. Brief description of the Drawings

Fig. 1 Effects of alpha-amylase and at least one lipase on starch loss from pasta made of T. aestivum or T. durum upon exposure to distilled water as determined via addition of Lugol’s solution. The images visualize the gradual starch loss from pasta in the presence of distilled water over time (0-60 min) at room temperature. Assessment of starch loss was based on the formation of dark inclusion compounds due to reaction of washed-out starch with Lugol’s solution, a) Pasta made of T. aestivum. First row: negative control, pasta without enzyme; second row: pasta comprising alpha-amylase; third row: pasta comprising the lipolytic enzyme Pastazym Super Flex; fourth row: pasta comprising another lipolytic enzyme, Pastazym Duo Pure, b) Pasta made of T. durum. First row: negative control pasta without enzyme; second row: pasta comprising alpha-amylase; third row: pasta comprising the lipolytic enzyme Pastazym Super Flex.

Fig. 2 Quantification of starch loss from Fig. 1 . The figure represents the calculated color differences between treated and untreated samples at different exposure times a) for pasta from soft wheat flour (T. aestivum), and b) for pasta from durum wheat semolina (T. durum). For precise calculation method, see example.

Fig. 3 Effects of alpha-amylase and at least one lipase on starch loss from pasta made of T. aestivum or T. durum upon exposure to distilled water as determined by assessing sample turbidity. The images display the turbidity caused by starch leakage into the soaking water from soft and hard wheat pasta samples after soaking for 20-60 min in distilled water at room temperature, a) Pasta made from T. aestivum. The first row shows an untreated reference sample, the second row the effect of fungal alpha-amylase, the third row the effect of the lipolytic enzyme Pastazym Super Flex, and the fourth row the effect of another lipolytic enzyme, Pastazym Duo Pure, b) Pasta made from T. durum. The first row shows an untreated reference sample, the second row the effect of fungal alpha-amylase, and the third row the effect of a lipolytic enzyme, Pastazym Super Flex.

Fig. 4 Effect of alpha-amylase or at least one lipase on the strength of pasta made from T. aestivum or T. durum upon prolonged exposure to distilled water at room temperature. The strength of the pasta structures was assessed after 30, 45 and 60 min using a Texture Analyzer. The graph in a) shows the results for pasta made of soft wheat (T. aestivum) flour, b) for pasta made of durum wheat (T. durum) semolina. Addition of the lipases Pastazym Super Flex or Pastazym Duo Pure slowed down the softening of soft wheat and durum wheat pasta in the presence of water, whereas amylase Alphamalt VC 5000 noticeably accelerated softening in both pasta types, as compared to the respective negative control pasta lacking any of these enzymes.

SEQ ID NO: 1 Amino acid sequence of lipase Pastazym Duo Pure

SEQ ID NO: 2 Amino acid sequence of lipase Pastazym Super Flex

SEQ ID NO: 3 Amino acid sequence of glucose oxidase Sternzym Gloxy (glucose oxidase from Saccharomyces cerevisiae, a FAD-linked glucose oxidase)

SEQ ID NO: 4 Amino acid sequence of Sternzym Gloxy TGO (glucose oxidase from Penicillium chrysogenum, a FAD-linked glucose oxidase)

SEQ ID NO: 5 Amino acid sequence of Thiolase, a FAD-linked sulfhydryl oxidase from Saccharomyces cerevisiae

SEQ ID NO: 6 Amino acid sequence of transglutaminase Sternzym PT 8001

SEQ ID NO: 7 Amino acid sequence of laccase Suberase

Example

In the following experiment, pasta was prepared with dough in the presence or absence of specific enzymes to assess changes in the stability and strength of the pasta in the presence of water. Starch loss is considered an indication of loss of stability, as the starch is washed out of the matrix. Accordingly, with increasing starch loss, the pasta becomes softer and less stable.

Materials and Methods

Preparation of pasta

For the pasta dough, 2,000 g of T. aestivum wheat flour or T. durum wheat semolina (for results of analyses see Table 1) were mixed with 520 mL or 600 mL tab water, respectively. The dough was formed into a tubular shape using laboratory-scale pasta press (MAC 30S, Italpast S.r.l., Italy). For the negative control group, the dough was prepared without adding any enzyme. In addition, pasta was prepared comprising 100 mg/kg of fungal alpha-amylase (Alphamalt VC 5000, Muhlenchemie GmbH & Co. KG, Germany), an enzyme capable of catalyzing the hydrolysis of starch. It is used e.g. for flour standardization and improvement. For the positive group, 150 mg/kg of lipolytic enzyme (Pastazym Duo Pure, or Pastazym Super Flex, Muhlenchemie GmbH & Co. KG, Germany) were added to the flour. Table 1: Analysis results for T. aestivum flour and T. durum semolina n.d. = not determined

UCD = Unites Chopin Dubois, Chopin Dubois units

FU = Farinograph units

In all trials, all dry ingredients, e.g. flour and enzymes, were premixed for 2 min at 118 mim ¹ in a Hobart N50 mixer (Hobart GmbH, Germany).

After forming, the resulting pasta structures were dried in a static dryer (Pavan, Italy) for

315 minutes at temperatures of up to 86 °C until a moisture content of the pasta of less than 12 % was reached. The drying was performed in 10 steps with different drying air temperature and moisture settings (see Table 2).

Table 2: Pasta drying process including air temperature, air moisture and time settings

Detection of starch loss from pasta

10 g of each pasta sample, either comprising 100 mg Pastazym Duo Pure or 60 mg Pas- tazym Super Flex per kg of flour/semolina or no enzyme at all, were added to a beaker containing 100 mL distilled water (22 °C). The amount of washed-out starch in the water was assessed after 20, 30, 45 or 60 min either by assessing the turbidity of the soaking water or, alternatively, after 0, 20, 30, 45 and 60 min by mixing 50 g of the soaking water with 10 drops of a 1 % (wt/wt) Lugol’s solution. Lugol’s solution intercalates into the a-helix of amyl- ose present in starch, resulting in the formation of dark inclusion compounds. The tests were performed at room temperature. Using a colorimeter (Chromameter CR-400/410, Kon- ica Minolta, Japan), the L*, a*, b* values were measured against a blank reference prepared by mixing 50 g distilled water with 10 drops of Lugol’s solution.

The L-value served as the main measure of brightness: The more starch in the solution, the lower the L-value, since the continuously increasing formation of black inclusion compounds reduced the brightness of the solution.

The color distance AE between the sample and the reference was subsequently calculated as follows:

Assessment of pasta strength

Strength of pasta was assessed after 30, 45 and 60 min with Texture Analyzer (TA.XT plus, Micro Stable Systems, USA) using 5 kg load cell. The force was measured with a Perspex blade (code A/LKB-F) as probe that penetrated three pasta tubes with defined path length. Therefore, the three pasta tubes were placed centrally under the Perspex knife on a HDP/90 Heavy Duty platform. With a test speed of 17 mm/s, the Perspex knife covered a total distance of 5.5 mm, starting at an initial height of 6 mm, while penetrating the three pasta tubes.

Results

Detection of starch loss from pasta made ofT. aestivum or T. durum

As shown in Fig. 1a and quantified in Tab. 3 and 4, the soaking water comprising pasta made from T. aestivum developed an increasingly darker coloration upon addition of Lugol’s solution as incubation time progressed, indicative of increased amounts of dissolved starch in the water. A similar observation was made when testing the soaking water containing pasta made of T. durum. However, in the absence of any enzyme, the loss of starch was more pronounced in pasta made of T. aestivum flour than that made of T. durum semolina. For both pasta types, the presence of alpha-amylase resulted in a noticeable increase of starch loss already after 20 to 30 min, as indicated by a much darker coloration of the water when mixed with Lugol’s solution. This was expected, as alpha amylase catalyzes the hydrolysis of starch and thus promotes the solubilization and hence the loss of starch into the surrounding medium. Addition of either Pastazym Super Flex or Pastazym Duo Pure to the pasta dough made of T. aestivum significantly alleviated the loss of starch from the pasta into the water, indicated by a lighter discoloration as compared to the reference (Fig. 1a and Fig. 2a). Similarly, the presence of Pastazym Duo Pure in the T. durum pasta sample significantly delayed starch loss from the pasta structure into the soaking water (Fig. 1b and 2b). These results were further confirmed when assessing starch loss from the different pasta samples by comparing the starch-induced turbidity of the soaking water after 20, 30, 45 or 60 min (Fig. 3). Of note, in pasta prepared from T. aestivum, the Pastazym Duo Pure appeared to prevent starch loss most effectively, as the Lugol’s solution-induced coloration as well as the turbidity of the soaking water of the pasta supplemented with this enzyme was significantly lower compared to that of the other samples (Fig. 2a and 3a, bottom row). Without being bound to theory we assume that the specific lipolytic activity of Pastazym Duo Pure is more suitable for prevention of starch losses because it is more specific for triglycerides, resulting in di- and monoglycerides and fatty acids with a higher affinity to starch than the lyso-lipids created by Pastazym Super Flex. The latter exerts hydrolytic activity also on glyco- and phospholipids, hence is less specific for triglycerides than Pastazym Duo Pure.

Table 3: Color values of untreated control samples

Table 4: Color values samples treated with Pastazym Duo Pure

5

Assessment of strength of pasta comprising T. aestivum or T. durum

The strength of pasta made with Pastazym Duo Pure or Pastazym Super Flex was compared to the strength of equivalent pasta structures without any enzyme (negative control). The pasta samples were soaked in water (22 °C) for 30, 45 and 60 minutes and subsequently subjected to mechanical stress using a measuring device. Both the enzyme-treated as well as the negative control pasta showed decreasing strength the longer the pasta was immersed in the water. However, the reduction in strength of pasta prepared from T. aestivum was less pronounced in the pasta comprising the Pastazym Duo Pure or Pastazym Super Flex, compared to the negative control (Fig. 4a). For pasta from durum wheat, only Pastazym Duo Pure was tested. Again, addition of the lipase resulted in improved strength as compared to the control pasta sample without any enzyme (Fig 4b). In both tests, fungal amylase decreased the pasta strength. Table 5 displays the quantified data for the stability assays as well as the relative stability of each sample, as determined by comparing each pasta sample to a respective reference sample without the enzymes, which had been soaked in water for the same duration. The table further underlines the positive effect of the lipases and the negative effect of the alpha-amylase on pasta strength.

Taken together, the enzyme Pastazym Duo Pure effectively stabilized both pasta structures made from either T. aestivum or T. durum, i.e . , the obtained pasta structures exhibited a comparable stability regardless of the wheat species used.

Therefore, it could be shown that by modifying pasta made of T. aestivum with a lipase, it was possible to increase its stability to a level higher than that of a conventional, commercially available pasta made of T. durum. The stabilizing effect of, e.g., a lipase thus allows for the use of the significantly cheaper T. aestivum wheat for the preparation of a highly stable food handling device, e.g., a drinking straw. In addition, it allows for further stabilizing food handling devices made from T. durum.

Table 5: Stability data for soft wheat and durum wheat pasta after soaking in distilled water

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