ENZYMATICALLY STABILIZED PASTA STRUCTURE AND METHOD OF PREPARING THE SAME

Abstract

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, ajar, 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.

Claims

1. (canceled)

2. 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.

3. The pasta structure of claim 2, wherein the enzyme is a carboxylester hydrolase.

4. The pasta structure of claim 2, wherein the carboxylester hydrolase is a lipase, preferably, having at least 80% amino acid sequence identity to SEQ ID NO: 1,

5. The pasta structure of claim 2, wherein the enzyme is an oxidase selected from the group comprising a glucose oxidase and a sulfhydryl oxidase.

6. The pasta structure of claim 2, wherein the enzyme is a transglutaminase.

7. The pasta structure of claim 2, wherein the enzyme is an oxygenase, optionally, a laccase.

8. The pasta structure of claim 2, wherein the enzyme is capable of reducing or preventing the loss of starch from the pasta when the structure is contacted with water.

9. The pasta structure of claim 2, wherein the enzyme is present in the dry pasta in a concentration of 1-500 mg/kg.

10. The pasta structure of claim 2, wherein the shaped structure is selected from the group comprising a straw, a bowl, a cup, a plate, a spoon, a fork, a knife, a jar, a funnel, a swizzle stick and a cone, preferably, a straw.

11. The pasta structure of claim 2, wherein the pasta comprises water and wheat selected from the group comprising Triticum aestivum, Triticum durum, Triticum dicoccum, Triticum spelta, Triticum monococcum and a mixture thereof, wherein the wheat preferably is Triticum aestivium.

12. The pasta structure of claims 2-11, wherein the wheat is provided as flour, semolina or a mixture thereof, preferably, flour.

13. The pasta structure of claim 2, wherein the pasta dough has an initial water content of 20-40% (wt/wt) prior to drying of the dough.

14. The pasta structure of claim 2, wherein the pasta is dry pasta, preferably having a water content of less than 14% (wt/wt), optionally, 5-14% (wt/wt).

15. A method of preparing the pasta structure of claim 2, 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.

16. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0084] 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.

[0085] 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.

[0086] 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.

[0087] 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.

[0088] SEQ ID NO: 1 Amino acid sequence of lipase Pastazym Duo Pure [0089] SEQ ID NO: 2 Amino acid sequence of lipase Pastazym Super Flex [0090] SEQ ID NO: 3 Amino acid sequence of glucose oxidase Sternzym Gloxy (glucose oxidase from Saccharomyces cerevisiae, a FAD-linked glucose oxidase) [0091] SEQ ID NO: 4 Amino acid sequence of Sternzym Gloxy TGO (glucose oxidase from Penicillium chrysogenum, a FAD-linked glucose oxidase) [0092] SEQ ID NO: 5 Amino acid sequence of Thiolase, a FAD-linked sulfhydryl oxidase from Saccharomyces cerevisiae [0093] SEQ ID NO: 6 Amino acid sequence of transglutaminase Sternzym PT 8001 [0094] SEQ ID NO: 7 Amino acid sequence of laccase Suberase

EXAMPLE

[0095] 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

[0096] 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, Mühlenchemie 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, Mühlenchemie GmbH & Co. KG, Germany) were added to the flour.

TABLE-US-00001 TABLE 1 Analysis results for T. aestivum flour and T. durum semolina Property Method Dimension Semolina Flour Moisture ICC 1190/1 % 11.6 14.1 Protein ICC 159 % 14.7 12.7 Wet gluten ICC 155 % 37.3 29.3 Gluten Index ICC 155 82 96 Falling Number ICC 107 s n.d. 332 Ash ICC 104/1 % 1.08 0.62 Starch damage ICC 172 %/UCD n.d. 4.94/13.9 Farinograph ICC 115/1 Water absorption % 59.1 54.9 Stability mm:ss 05:51 08:31 Softening (12 min) FU 56 53 n.d. = not determined UCD = Unités Chopin Dubois, Chopin Dubois units FU = Farinograph units

[0097] In all trials, all dry ingredients, e.g. flour and enzymes, were premixed for 2 min at 118 min.sup.−1 in a Hobart N50 mixer (Hobart GmbH, Germany).

[0098] 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-US-00002 TABLE 2 Pasta drying process including air temperature, air moisture and time settings Step Temperature (° C.) Air humidity (%) Duration (min) 1 68 50 10 2 68 65 15 3 75 70 20 4 85 75 30 5 85 78 35 6 86 75 50 7 79 79 45 8 77 79 45 9 75 70 45 10 25 55 20

Detection of Starch Loss from Pasta

[0099] 10 g of each pasta sample, either comprising 100 mg Pastazym Duo Pure or 60 mg Pastazym 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 amylose 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, Konica 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.

[0100] 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.

[0101] The color distance ΔE between the sample and the reference was subsequently calculated as follows:

ΔE*=√{square root over ((ΔL*).sup.2+(Δa*).sup.2+(Δb*).sup.2)}

Assessment of Pasta Strength

[0102] 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 of T. aestivum or T. durum

[0103] 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 (FIGS. 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 (FIGS. 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-US-00003 TABLE 3 Color values of untreated control samples Time (min) Standard (no enzyme) 0 20 30 45 60 L* 67.06 46.53 45.07 41.30 39.23 a* −1.95 2.39 2.71 2.98 3.07 b* 29.94 10.76 9.28 0.88 −0.63 Color distance ΔE* 0.00 23.58 24.32 26.24 28.29

TABLE-US-00004 Color distances Time (min) ΔL* Δa* Δb* 0 0 0 0 20 20.53 4.34 19.18 30 21.99 4.66 20.66 45 25.76 4.93 29.07 60 27.83 5.02 30.57

TABLE-US-00005 TABLE 4 Color values samples treated with Pastazym Duo Pure Time (min) Enzyme treated 0 20 30 45 60 L* 67.06 61.04 57.13 56.81 55.58 a* −1.95 0.06 0.12 0.17 0.35 b* 29.94 22.83 18.65 12.56 10.11 Color distance ΔE* 0.00 9.53 15.18 20.29 23.03

TABLE-US-00006 Color distances Time (min) ΔL* Δa* Δb* 0 0 0 0 20 6.02 2.01 7.11 30 9.93 2.07 11.29 45 10.25 2.12 17.38 60 11.48 2.30 19.83

Assessment of Strength of Pasta Comprising T. aestivum or T. durum

[0104] 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.

[0105] 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.

[0106] 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-US-00007 TABLE 5 Stability data for soft wheat and durum wheat pasta after soaking in distilled water Stability (g) Soaking time (min) Sample 30 45 60 Soft wheat pasta Standard, untreated 1278 955 819 Alphamalt VC 5000, 100 ppm 1177 947 778 Pastazym Super Flex, 60 ppm 1343 1131 904 Pastazym Duo Pure, 100 ppm 1425 1015 861 Durum wheat pasta Standard, untreated 1346 1017 940 Alphamalt VC 5000, 100 ppm 1237 986 926 Pastazym Duo Pure, 100 ppm 1557 1150 1043

TABLE-US-00008 Relative stability (%), compared to standard after same soaking time Soaking time (min) Sample 30 45 60 Soft wheat pasta Standard, untreated 100 100 100 Alphamalt VC 5000, 100 ppm 92 99 95 Pastazym Super Flex, 60 ppm 105 118 110 Pastazym Duo Pure, 100 ppm 112 106 105 Durum wheat pasta Standard, untreated 100 100 100 Alphamalt VC 5000, 100 ppm 92 97 99 Pastazym Duo Pure, 100 ppm 116 113 111

REFERENCES

[0107] Kane et al., 2020, Seafloor microplastic hotspots controlled by deep-sea circulation. Science 368(6495), 1140-1145.

[0108] AU 2018101026

[0109] WO 2020/069587

[0110] WO2020/044049

[0111] Zanini de Vita, O., 2009. Encyclopedia of pasta, Volume 26, University of California Press

[0112] Zilic et al., 2011. Characterization of proteins from grain of different bread and durum wheat genotypes. Int. J. Mol. Sci. 12(9), 5878-5894.

[0113] Shewry et al., 2002. The structure and properties of gluten: an elastic protein from wheat grain. Phil. Trans. R. Soc. Lond. 357, 133-142.

[0114] Sacchetti et al., 2011. Effect of semolina particle size on the cooking kinetics and quality of spaghetti. Proc. Food Sci. 1, 1740-1745.

[0115] 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.

[0116] Horstmann et al., 2017 Starch characteristics linked to gluten-free products. Foods 6(4), 29-50.

[0117] Kibar et al., 2014. Effects of fatty acid addition on the physicochemical properties of corn starch. Int. J. Food Prop. 17(1), 204-218.

[0118] Morrison, W. R., 1994. Wheat lipids: structure and functionality. Bushuk W., Rasper V. F. (eds) Wheat. Springer.

[0119] 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.

[0120] Selinheimo, E., 2008. Tyrosinase and laccase as novel crosslinking tools for food biopolymers, PhD Thesis, VTT publications, 693.

[0121] www.europarl.europa.eu/news/en/headlines/society/20181212STO21610/plastic-waste-and-recycling-in-the-eu-facts-and-figures

[0122] www.bund.net/fileadmin/user_upload_bund/publikationen/chemie/chemie_plastikatlas_2019.pdf

[0123] www.wisefood.eu

[0124] makkaroni-strohhalme-kaufen.de

[0125] www.sausalitos.de/shop/sausaroni-pasta-strohhalme˜p44976

[0126] www.vomfass.de/pasta-strohhalme

[0127] www.pastastraws.org

[0128] stroodles.co.uk

[0129] www.drinkstuff.com/products/product.asp?ID=27441

[0130] en.wikipedia.org/wiki/Semolina

[0131] www.haverparticleanalysis.com/en/sieve-analysis/ro-tapr-test-sieve-shaker/

[0132] www.retsch.com/products/sieving/sieve-shakers/as-200-control/function-features/

[0133] gkm-net.de/en/laboratory-air-jet-lab-sieves.html

[0134] www.prvhh.de/newsroom/muehlenchemie/de/pressemeldung/news/detail/News/pastazym-pd-erzielt-starke-verbesserung-der-kochtoleranz-von-pasta/

[0135] texturetechnologies.com/industries/food-texture-analysis/pasta#:˜:text=XTPlus%20Texture%20Analyzer%20is%20accurate,will%20accept%20your%20test%20results