Modified gelatinized potato starch

Abstract

Modified potato starch composition obtained or available through a process comprising the following steps: f) Providing gelatinized potato starch; g) Providing one or more alpha amylases selected from the group consisting of enzyme species Genebank deposit number To uncultured marine bacterium ADK21254.1 Ac Anaerocolumna cellulosilytica WP_197978574.1 Po Petrotoga mobilis WP_012208959.1 h) Incubating the gelatinized potato starch from step a) with one or more alpha-amylases from step b), for example for a period of 5 - 180 minutes at a temperature in the range of 40 - 70°C; i) optionally inactivating one or more alpha-amylases; and j) Providing a modified potato starch composition.

Description

Technical field of the invention

[0001] The present invention relates to modified gelatinized potato starches. In particular, the present invention relates to modified potato starches produced by modification with one or more alpha amylases from the group consisting of Anaerocolumna cellulosilytica (Ac, WP_197978574.1), Petrotoga mobilis (Po, WP_012208959.1), and an uncultured marine bacterium (Um, ADK21254.1), as defined, for example, in SEQ ID NOs:1-3. The present invention also relates to processes for producing modified starches, uses of such modified starches, and food products comprising such modified starches. Background of the invention

[0002] Starch, a carbohydrate widely found in nature and a major energy reserve in many plants, consists of two types of molecules: amylose and amylopectin. Both polymers are composed of repeating units of D-glucose linked primarily by 1,4-α-glycosidic bonds. Amylopectin is highly branched by additional 1,6-α-glycosidic bonds, while amylose is mostly linear with few branching points.

[0003] In the past, starch modifications were primarily achieved through chemical derivatization, with acid hydrolysis being the most common method. Starches produced by acid hydrolysis are widely used as stabilizers and texturizing agents in processed foods. Due to their exceptional gelling properties, these starches are increasingly preferred as food ingredients, either partially or completely replacing animal-based ingredients in products such as confectionery and dairy. Recently, there has been growing interest in the enzymatic modification of starch as a "clean-label" alternative to conventional chemical processes like acid hydrolysis.Unlike their chemically modified counterparts, enzyme-modified starches are classified as ingredients rather than additives, reflecting the growing consumer demand for simpler and more transparent food products. Furthermore, the production of chemically modified starches generates a relatively large quantity of inorganic salts, which must be managed to prevent their release into surrounding water sources. Consequently, research efforts are focused on developing enzymatic modification techniques to produce modified starches with a range of diverse functional properties tailored to specific applications in the food industry.

[0004] Microbial α-amylases (EC 3.2.1.1) are among the most widely used enzymes in the starch processing industry. These enzymes enable the depolymerization of starch by catalyzing the endolytic hydrolysis of 1,4 α-bonds in amylose and amylopectin. Most α-amylases are classified as glycoside hydrolases in family 13 (GH13), although some α-amylase-specific enzymes are found in other families such as GH57, GH119, and GH126. However, the majority of commercially available α-amylases are developed and used for the complete breakdown of starch, for example, to glucose syrup, and for bioethanol production.

[0005] Zinck et al. (Molecules 2023, 28, 2947) demonstrate the importance of the inactivation process in the alpha-amylase-mediated depolymerization of crude starch and provide a method for efficient alpha-amylase inactivation in starch systems. The α-amylase Um-αAmy (AEM89278.1 / ADK21254.1) was tested on crude potato starch.

[0006] Liu et al. (Mar Biotechnol (2012) 14:253-260) reveals the identification and phylogenetic characterization of a new subfamily of an α-amylase enzyme from marine microorganisms.

[0007] Therefore, an improved process for producing modified starches would be advantageous, and in particular, more efficient and / or reliable starch compositions with unique properties would be beneficial. Summary of the invention

[0008] The present invention relates to the enzymatic modification of gelatinized potato starch using α-amylases to provide starches with improved properties, such as enhanced gelling and / or texturizing properties. Three α-amylases have been identified that are particularly suitable for modifying gelatinized potato starch. The resulting enzymatically modified potato starches are considered useful for the food industry, for example, as gelling agents and texturizers. The enzymatically modified gelatinized potato starches may be particularly suitable as gelling agents in cheese analogues and gummy candies.

[0009] The tested amylases originate from Anaerocolumna cellulosilytica (Ac, WP_197978574.1), Petrotogo mobilis (Po, WP_012208959.1) and an uncultured marine bacterium (Um, ADK21254.1).

[0010] Example 2 shows that gelatinized potato starches modified with the Um, Ac, and Po enzymes exhibit much better gelling properties and higher stability compared to potato starches modified with commercially available α-amylases. Example 3 shows that starch solutions based on gelatinized potato starches modified with the Ac, Po, and Um enzymes show a much lower tendency to release water after storage (lower syneresis) compared to potato starches modified with commercially available α-amylases.

[0011] Example 4 shows that the enzymatically modified gelatinized potato starches obtained with the enzymes Ac, Po, and Um exhibit different molecular weight distribution profiles compared to tested commercially available amylases. Without being bound to a specific theory, it is assumed that this difference in molecular weight distribution profiles contributes to the differences in functional properties described in Examples 2 and 3.

[0012] Example 5 shows that the enzymatically modified gelatinized potato starches obtained with the enzymes Ac, Po, and Um exhibit different chain length distribution profiles compared to tested commercially available amylases. Without being bound to a specific theory, it is assumed that this difference in chain length distribution profiles contributes to the differences in functional properties described in Examples 2 and 3.

[0013] One object of the present invention is to provide an improved method for producing modified potato starches with unique properties. In particular, one object of the present invention is to provide modified potato starches that exhibit improved gelling and / or texturizing and / or syneresis properties.

[0014] Thus, one aspect of the invention relates to a method for producing a modified potato starch composition, wherein the method comprises: a) Providing a gelatinized potato starch; b) Providing one or more alpha amylases selected from the group consisting of enzyme species Genebank deposit number To uncultured marine bacterium ADK21254.1 Ac Anaerocolumnacellulosilytica WP_197978574.1 Po Petrotoga mobilis WP_012208959.1 c) Incubating the gelatinized potato starch from step a) with one or more alpha-amylases from step b), for example for a period of 5 - 180 minutes at a temperature in the range of 40 - 70°C; d) if necessary, inactivating one or more alpha-amylases; and e) Providing a modified potato starch composition.

[0015] Another aspect of the present invention relates to a modified potato starch composition which is obtained or available by a method according to the invention.

[0016] Another aspect of the present invention relates to the use of an alpha-amylase selected from the group consisting of enzyme species Genebank deposit number To uncultured marine bacterium ADK21254.1 Ac Anaerocolumnacellulosilytica WP_197978574.1 Po Petrotoga mobilis WP_012208959.1 to modify a gelatinized potato starch.

[0017] Another aspect of the present invention is to provide a modified gelatinized potato starch composition which has a ratio of the degree of glucose polymerization of %Total(DP1+DP6)%Total(DP2−5+DP7−10) above 0.32, e.g. above 0.34; and / or a ratio of the degree of glucose polymerization of %Total(DP1+DP6)%Total(DP2−5+DP7−10) in the range 0.33 - 0.7, e.g. in the range 0.35 - 0.7; preferably in the range 0.35 - 0.6; and / or - a glucose polymerization degree % of total (DP6 -10) below 11.4; and / or - a glucose polymerization degree % of total (DP6 - 10) in the range 9 - 11.3, e.g. 9.2 - 11, e.g. 9.5 - 10; and / or - a molecular weight distribution (R) below 0.6; e.g. in the range 0.2 - 0.59, preferably in the range 0.25 - 0.4; and more preferably in the range 0.25 - 0.35; where the molecular weight distribution (R) is defined as R = ∫ medium molecular weight power fraction ∫ low molecular weight power fraction + ∫ high molecular weight power fraction Low molecular weight starch fraction: Mw = 5.0 * 10 2 - 3.3 * 10 4 Since the mean molecular weight of the starch fraction is: Mw = 3.4 * 10 4 - 8.9 *105 Since the starch fraction has a high molecular weight: Mw = 9.0 * 10 5 - 4.0 *10 7 There.

[0018] Another aspect of the present invention relates to a food ingredient or food product comprising the potato starch composition according to the invention.

[0019] Another aspect of the invention relates to the use of a modified potato starch according to the invention as a gelling agent and / or texturizer, such as for gelling cheese, such as a normal cheese or a cheese analogue, for gelling gummy candies or as a gelatin substitute.

[0020] It is assumed that the process according to the invention is also suitable for other types of starch besides the gelatinized potato starch defined above. Thus, in an alternative aspect of the invention, in step a), the gelatinized starch is any starch and / or a starch of any origin, wherein the starch is, for example, selected from the group consisting of tapioca starch, corn starch, wheat starch, barley starch, rice starch, pea starch, sweet potato starch, bean starch, arrowroot starch, millet starch, kudzu starch (and probably many others). It is understood that these aspects of the invention can also be combined with other aspects, points, and embodiments of the invention. Brief description of the characters Fig. 1 Fig. Figure 1 shows the procedure for evaluating the syneresis of a starch solution. Fig. 2 Fig.Figure 2 shows gelled starches after 2 days at 5°C. Top right and left: Starch #1 (unmodified). Bottom right and left: Starch #6 (modified with Spezyme FRED). Fig. 3 Fig. Figure 3 shows the syneresis of starch solutions #4 (Ac), #12 (Po) and #14 (GC007) after storage at 5°C for 7 days. Fig. 4 Fig. Figure 4 shows the syneresis of starch solutions #17 (Um) and #20 (specyme endurase) after storage at 5°C for 14 days. Fig. 5 Fig. Figure 5 shows the molecular weight distribution of the enzymatically modified gelatinized starches #1 (Um - reaction time 5 minutes), #2 (Um - reaction time 40 minutes) and #6 (Spezyme FRED - reaction time 20 minutes). Fig. 6 Fig. Figure 6 shows the molecular weight distribution of the enzymatically modified gelatinized starches #3 (Ac - reaction time 5 minutes), #4 (Ac - reaction time 60 minutes), #5 (Po - reaction time 40 minutes) and #7 (GC007 - reaction time 40 minutes). Fig. 7 Fig. Figure 7 shows the change in the chain length distribution of the enzymatically modified starches #1 (Um - reaction time 5 minutes) and #2 (Um - reaction time 40 minutes). Fig. 8 Fig. Figure 8 shows the change in the chain length distribution of the enzymatically modified starch #6 (specyme FRED - reaction time 20 minutes). Fig. 9 Fig. Figure 9 shows the change in the chain length distribution of the enzymatically modified starches #3 (Ac - reaction time 5 minutes) and #4 (Ac - reaction time 60 minutes). Fig. 10 Fig. Figure 10 shows the change in the chain length distribution of the enzymatically modified starch #5 (Po - reaction time 40 minutes). Fig. 11 Fig. Figure 11 shows the change in the chain length distribution of the enzymatically modified starch #7 (GC007 - reaction time 40 minutes).

[0021] The present invention will now be described in detail below. Detailed description of the invention Definitions

[0022] Before the present invention is explained in detail, the following terms and conventions will first be defined: Gelatinized starch

[0023] Gelatinized (potato) starch is a starch that has been preheated in water and swollen by absorbing water.

[0024] Gelatinization occurs when starch granules are heated in water, causing them to swell and absorb water. This process breaks the intermolecular bonds of the starch molecules, making them more accessible to enzymes and increasing their digestibility. Furthermore, the Maltese cross pattern of starch granules visible under a polarizing microscope is absent in gelatinized starch due to the loss of crystallinity. Gelatinized starch becomes viscous and develops a gel-like consistency, depending on the amount of starch present.

[0025] Gelatinized starch is frequently used in cooking to thicken sauces, soups, and other food products, and for various applications in foods where a thickening or gelling agent is needed. Examples include gravies, puddings, and pie fillings.

[0026] An example of how gelatinized (potato) starches are produced is shown in Example 1. Those skilled in the art are aware of other processes for producing gelatinized starches. Pregelatinized starch

[0027] Pregelatinized starch is precooked (gelatinized) and then dried. This makes it instantly soluble in cold or hot water. The cooking and drying steps can be carried out in several stages or simultaneously. Examples include drum drying, extrusion, or jet cooking followed by spray drying. However, those skilled in the art are also aware of other methods for producing pregelatinized starches. Soluble starch

[0028] Soluble starch is granular starch that has been physically modified to alter its solubility. This can be achieved through an alcoholic-alkaline treatment. The starch should be soluble in cold water; however, manufacturers recommend adding a small amount of cold water followed by boiling water, which calls into question the actual solubility of these starches in cold water. Unlike gelatinized and pregelatinized starches, the starch granules retain their Maltese cross pattern under a polarizing microscope. Raw starch

[0029] Raw starch refers to starch in its natural, untreated form that has not been gelatinized or partially gelatinized. Syneresis

[0030] Syneresis is the extraction or displacement of a liquid from a gel. A method for determining the degree of syneresis, as used herein, is described in Example 1. degree of glucose polymerization

[0031] The degree of polymerization (DP) of glucose units is used as a measure of chain length in starch molecules. These chain lengths are determined after the breaking of the 1,6-α bonds, as further described in Example 1. Ratio of molecular weight distribution (R)

[0032] The ratio of the molecular weight distribution (R) between the medium molecular weight starch molecule fraction and the low and high molecular weight starch molecule fraction can be calculated for any enzymatically modified starch. The ratio (R) is expressed as follows: R = ∫ medium molecular weight power fraction ∫ low molecular weight power fraction + ∫ high molecular weight power fraction Low molecular weight starch fraction: Mw = 5.0 * 10 2 - 3.3 * 10 4Since the mean molecular weight of the starch fraction is: Mw = 3.4 * 10 4 - 8.9 *10 5 Since the starch fraction has a high molecular weight: Mw = 9.0 * 10 5 - 4.0 *10 7 There.

[0033] Further details can be found, for example, in examples 1 and 4. Cheese analogues

[0034] In this context, "cheese analogue" or "alternative cheese" refers to products used as culinary substitutes for cheese. These are typically products made by blending other fats or proteins and used in prepared foods. This category of cheese includes vegan cheeses as well as some dairy-based products, such as processed cheese, which are not considered traditional cheeses. These foods may be intended as substitutes for cheese, as with vegan products, or as alternatives, such as products used in salad bars and pizza making, which may have different properties, such as lower cholesterol or different melting points. Gummy candies / wine jam

[0035] Gummy candies or wine gums typically contain a sugar base, colorings, flavorings, and a gelling agent (hydrocolloid). The most commonly used gelling agent is gelatin. Of course, sugar-free alternatives also exist. Examples of gelling agents or hydrocolloids include carrageenan, agar, xanthan gum, locust bean gum, gellan gum, gum arabic, pectin, gelatin, kappa-carrageenan, guar gum, and modified or unmodified starches.

[0036] The starch composition according to the present invention can replace any of the gelling agents or hydrocolloids above, in particular gelatin.

[0037] In the present context, the terms “gummy candies”, “wine gums”, “jelly candies” or “gummy sweets” are used interchangeably as examples of gelled foodstuffs according to the present invention. gelatin

[0038] Gelatin is a collection of peptides and proteins produced by the partial hydrolysis of collagen, which is extracted from the skin, bones, and connective tissue of animals such as cattle, chickens, pigs, and fish. Gelatin is a well-known gelling agent, for example, in the production of gummy candies. Sequence identity

[0039] In the context of the present invention, the terms "sequence identity" or "homologous" denote a quantitative measure of the degree of homology between two amino acid sequences or between two nucleic acid sequences. If two sequences to be compared do not have the same length, they must be aligned to fit together as closely as possible, whereby the insertion of gaps or, alternatively, truncation at the ends of the polypeptide or nucleotide sequences is possible. Sequence identity can be expressed as (Nref−Ndif)100Nref are calculated, where N dif The total number of non-identical residues in the two sequences, when aligned, is N ref The number of residues in one of the sequences is therefore . The DNA sequence AGTCAGTC will have a sequence identity of 75% (N) with the sequence AATCAATC. dif = 2 and N ref = 8). A gap is considered a non-identity of the specific residue(s), i.e., the DNA sequence AGTGTC will have a sequence identity of 75% (N) with the DNA sequence AGTCAGTC. dif = 2 and N ref = 8).

[0040] In connection with all embodiments of the invention relating to amino acid or nucleotide sequences, the percentage of sequence identity between one or more sequences can also be based on alignments using the software clustalW (http: / / www.ebi.ac.uk / clustalW / index.html) with default settings. For nucleotide sequence alignment, these settings are: Alignment=3Dfull, Gap Open 10.00, Gap Ext. 0.20, Gap Separation Dist. 4, DNA weight matrix: Identity (IUB). For amino acid sequence alignment, these settings are: Alignment=3Dfull, Gap Open 10.00, Gap Ext. 0.20, Gap Separation Dist. 4, Protein weight matrix: Gonnet.

[0041] Alternatively, nucleotide sequences can be analyzed using the DNASIS Max program, and the sequences can be compared at http: / / www.paralign.org / . This service is based on the two comparison algorithms Smith-Waterman (SW) and ParAlign. The first algorithm was published by Smith and Waterman (1981) and is an established method for determining the optimal local alignment of two sequences. The other algorithm, ParAlign, is a heuristic method for sequence alignment; details of the method are published in Rognes (2001). Default settings for the score matrix, gap penalties, and E-values ​​were used. Method for producing a modified potato starch composition

[0042] As also described above, part of the present invention relates to the enzymatic modification of gelatinized potato starch using unique α-amylases to provide starches with improved properties, such as enhanced gelling and / or texturizing properties. Three α-amylases have been identified that are particularly suitable for modifying gelatinized potato starch. Thus, a first aspect of the invention relates to a method for producing a modified potato starch composition, the method comprising: a) Providing a gelatinized potato starch; b) Providing one or more alpha amylases selected from the group consisting of enzyme species Genebank deposit number To uncultured marine bacterium ADK21254.1 Ac Anaerocolumnacellulosilytica WP_197978574.1 Po Petrotoga mobilis WP_012208959.1 c) Incubating the gelatinized potato starch from step a) with one or more alpha-amylases from step b), for example for a period of 5 - 180 minutes at a temperature in the range of 40 - 70°C; d) if necessary, inactivating one or more alpha-amylases; and e) Providing a modified potato starch composition.

[0043] As shown in examples 2 and 3, the provided modified starch compositions exhibit unique gelling properties and lower syneresis. Step a)

[0044] The gelatinized potato starch can be provided from various sources. In one embodiment, in step a), the gelatinized potato starch is selected from the group consisting of a gelatinized native potato starch, a gelatinized chemically modified potato starch, a gelatinized enzymatically modified potato starch, and a gelatinized physically modified potato starch, or combinations thereof. Preferably, the gelatinized potato starch is a gelatinized native potato starch. In a further embodiment, in step a) - The gelatinized potato starch is selected from the group consisting of waxy potato starch, potato starch with an amylose content of over 30% by weight; and / or - is the gelatinized chemically modified potato starch selected from the group consisting of acid-hydrolyzed starch, succinate starch such as octenylsuccinate starch, hydroxypropyl starch, oxidized starch, cross-linked starch such as diphosphate starch or adipic acid-cross-linked starch, acetylated starch and phosphorylated starch; and / or - The gelatinized physically modified starch is selected from the group consisting of heat-treated starches such as dry-heated starch, heat-moisture-treated starch and tempered starch, pressure-treated starch, extruded starch, drum-dried starch, spray-dried starch, radiation-treated starch such as ultrasonically treated starch and microwave-treated starch; and / or - The gelatinized enzymatically modified potato starch is selected from the group consisting of starches modified with glycoside hydrolases such as other α-amylases, beta-amylase, glucoamylases, maltogenic alpha-amylases and branching enzymes (e.g., pullulanase and isoamylase), glycosyltransferases such as 4-alpha-glucanotransferases, branching enzymes and cyclodextrin glucanotransferases.

[0045] In the present context, a “wax starch” is a starch with an amylopectin content of more than 90 wt.%, such as more than 95 wt.%, such as more than 99 wt.% amylopectin content of the starch.

[0046] In the present context, an “amylose-rich starch” is a starch with an amylose content of more than 30% by weight.

[0047] Gelatinized potato starch can be produced in various ways. In one embodiment, the gelatinized potato starch from step a) is produced using a process selected from the group consisting of heating to at least 60°C, preferably at least 80°C, more preferably at least 95°C, such as heating in the presence or absence of cations such as CaCl2. Step b)

[0048] In one embodiment, the one or more alpha-amylases are defined by - an amino acid sequence selected from the group consisting of SEQ ID NO:1 (Ac), SEQ ID NO:2 (Po) and SEQ ID NO:3 (Um), or - an amino acid sequence with at least 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO:1 (Ac), SEQ ID NO:2 (Po) and SEQ ID NO:3 (Um), such as at least 95% sequence identity, e.g. 98% sequence identity or e.g. 99% sequence identity.

[0049] As explained further below in the section on sequences, SEQ ID NOs:1-3 are listed without signal peptides. Signal peptides can be added, e.g., N-terminal. SEQ ID NO:4 is the wt signal peptide for Ac, SEQ ID NO:5 is the wt signal peptide for Po, and SEQ ID NO:6 is an example His tag.

[0050] Thus, in one embodiment, the alpha-amylases comprise a signal peptide or an affinity tag, preferably N-terminal, e.g., selected from SEQ ID NOs:4-6. In another embodiment, the affinity tag is a His tag, e.g., SEQ ID NO:6. A person skilled in the art can attach other types of affinity tags. Step c)

[0051] It is possible to use different amounts / concentrations of enzymes, however, the specific amounts should be titrated, e.g. also based on temperature and time, but also on the enzyme batch.

[0052] In one embodiment, the temperature during step c) is in the range of 20 to 90°C, e.g. 20 to 40°C, e.g. 40 to 60°C or e.g. 70 to 90°C.

[0053] For the enzymes according to the invention, the following temperatures are considered optimal: • The optimum for Ac is between 40-60 °C. • The optimum temperature for Po is 70-90 °C. • The optimum temperature for Um is between 20-40 °C.

[0054] In another embodiment, step c) is carried out over a period of time ranging from 5 minutes to 120 minutes, e.g. 5 minutes to 60 minutes.

[0055] For example, the expert can adjust the enzyme concentration, temperature and / or reaction time to achieve a desired overall degree of starch degradation. Step d)

[0056] The enzymes are preferably inactivated after step c). Inactivation can be carried out in many different ways. In one embodiment, one or more alpha-amylases are selected from the group consisting of using a method. • NaOCl treatment, preferably followed by reduction using NaHSO3; • Heat inactivation, such as heating to at least 90°C; • low pH value, such as below pH 5, such as below pH 3; • high pH value, such as above pH 10, such as above pH 12 inactivated. Step e)

[0057] The modified starch compositions obtained in step e) may exhibit unique structural features. For example, in one embodiment, the potato starch composition provided is a starch composition according to the invention.

[0058] The provided starch can also be in a dry form. Thus, in one embodiment, step e) comprises the following steps: - e1) Drying the starch; and - e2) Providing a dried modified potato starch composition.

[0059] In a further embodiment, the drying step e1) is carried out using a method selected from the group consisting of spray drying, flash drying, drum drying and freeze drying.

[0060] In yet another embodiment, the dried modified potato starch composition has a water content of less than 20 wt.%, preferably less than 15 wt.%, more preferably less than 10 wt.% water.

[0061] In another embodiment, the starch in the provided potato starch composition has: I) - a ratio of the degree of glucose polymerization of %Total(DP1+DP6)%Total(DP2−5+DP7−10) above 0.32, e.g. above 0.34; and / or - a ratio of the degree of glucose polymerization of %Total(DP1+DP6)%Total(DP2−5+DP7−10) in the range 0.33 - 0.7, e.g. in the range 0.35 - 0.7; preferably in the range 0.35 - 0.6; and / or II) - a glucose polymerization degree % of total (DP6 - 10) below 11.4; and / or - a glucose polymerization degree % of total (DP6 - 10) in the range 9 - 11.3, e.g. 9.2 - 11, e.g. 9.5 - 10; and / or III) Δ%Total(DP1−5)Δ%Total(DP6−10)>1, preferably above 1.5, and / or Δ%Total(DP1−5)Δ%Total(DP6−10) in the range 1 - 2.5, preferably in the range 1.5 - 2.3, more preferably in the range 1.7 - 2.2; where Δ% of total is calculated as a comparison with a corresponding gelatinized potato starch before enzymatic modification; and / or IV) - a molecular weight distribution (R) below 0.6; e.g. in the range 0.2 - 0.59, preferably in the range 0.25 - 0.4; and more preferably in the range 0.25 - 0.35; where the molecular weight distribution (R) is defined as R = ∫ medium molecular weight power fraction ∫ low molecular weight power fraction + ∫ high molecular weight power fraction Low molecular weight starch fraction: Mw = 5.0 * 10 2 - 3.3 * 10 4 Since the mean molecular weight of the starch fraction is: Mw = 3.4 * 10 4 - 8.9 *10 5 Since the starch fraction has a high molecular weight: Mw = 9.0 * 10 5 - 4.0 *10 7 There.

[0062] As shown in Examples 4 and 5, these unique structural features of the obtained starch compositions were identified by careful structural analysis and comparisons with modified starch compositions obtained using commercially available enzymes. Product-by-Process

[0063] In another aspect, the invention relates to a modified potato starch composition obtained or available by a process according to the invention. As shown in Examples 2 and 3, the starch compositions obtained have unique properties compared to commercially available alternatives. Uses

[0064] In another aspect, the invention relates to the use of an alpha-amylase selected from the group consisting of enzyme species Genebank deposit number To uncultured marine bacterium ADK21254.1 Ac Anaerocolumnacellulosilytica WP_197978574.1 Po Petrotoga mobilis WP_012208959.1 to modify gelatinized potato starch.

[0065] In one embodiment, the use serves to produce a modified starch with improved gelling properties and / or texturizing properties and / or lower syneresis.

[0066] In another embodiment, the improved gelling properties are increased hardness and / or higher complex modulus. Modified gelatinized potato starch compositions

[0067] The present invention also relates to modified gelatinized potato starch compositions. As shown, for example, in Examples 4 and 5, specific structural features for the modified starch compositions have been identified.

[0068] Thus, one aspect of the invention relates to a modified gelatinized potato starch composition which has: - a ratio of the degree of glucose polymerization of %Total(DP1+DP6)%Total(DP2−5+DP7−10) above 0.32, e.g. above 0.34; and / or - a ratio of the degree of glucose polymerization of %Total(DP1+DP6)%Total(DP2−5+DP7−10) in the range 0.33 - 0.7, e.g. in the range 0.35 - 0.7; preferably in the range 0.35 - 0.6.

[0069] As shown in Example 5 (see Table 12a-b), such ratios of glucose polymerization degree are not found in the batches of starch modified with commercially available amylases. In particular, the starches modified with Ac and Po stand out with regard to the ratio of their polymerization degrees.

[0070] In another aspect, the invention relates to a modified gelatinized potato starch composition which comprises: - Δ%Total(DP1−5)Δ%Total(DP6−10)>1, preferably above 1.5, and / or - Δ%Total(DP1−5)Δ%Total(DP6−10) in the range 1 - 2.5, preferably in the range 1.5 - 2.3, more preferably in the range 1.7 - 2.2; where Δ% total is calculated as a comparison with a corresponding gelatinized potato starch before enzymatic modification.

[0071] As shown in Example 5 (see Table 11a-b), such ΔDP ratios of glucose polymerization degree are not found in the batches of starch modified with commercially available amylases. In particular, the starches modified with Um stand out with respect to such ΔDP ratios.

[0072] In yet another aspect of the invention, the modified gelatinized potato starch composition according to the invention has a molecular weight distribution (R) of less than 0.6; e.g., in the range of 0.2 - 0.59, preferably in the range of 0.25 - 0.4; and more preferably in the range of 0.25 - 0.35; wherein the molecular weight distribution (R) is defined as R = ∫ medium molecular weight power fraction ∫ low molecular weight power fraction + ∫ high molecular weight power fraction Low molecular weight starch fraction: Mw = 5.0 * 10 2 - 3.3 * 10 4 Since the mean molecular weight of the starch fraction is: Mw = 3.4 * 10 4- 8.9 *10 5 Since the starch fraction has a high molecular weight: Mw = 9.0 * 10 5 - 4.0 *10 7 There.

[0073] In one embodiment, the molecular weight (Mw) of the starch is determined as described in Example 1.

[0074] As shown in Example 4 (see, for example, Table 10a-b), such a molecular weight (Mw) distribution of starch is not found in batches of starch modified with commercially available amylases. In particular, the enzymes according to the invention provide values ​​that are lower than the values ​​observed with commercially tested enzymes.

[0075] The modified gelatinized potato starch composition exhibits yet another aspect of the invention: - a glucose polymerization degree % of total (DP6 - 10) below 11.4; and / or - a glucose polymerization degree % of total (DP6 - 10) in the range 9 - 11.3, e.g. 9.2 - 11, e.g. 9.5 - 10.

[0076] As shown in Example 5 (see Tables 13a-b), the starch composition modified with "Um" in particular exhibits such low values. In one embodiment, such a composition was modified with or includes Um, such as Um in its inactivated state.

[0077] In one embodiment, the modified gelatinized potato starch composition comprises one or more enzymes selected from: enzyme species Genebank deposit number To uncultured marine bacterium ADK21254.1 Ac Anaerocolumnacellulosilytica WP_197978574.1 Po Petrotoga mobilis WP_012208959.1 Preferably, one or more enzymes are inactivated.

[0078] If the final starch composition is not purified, small amounts of the enzymes may be present in the composition, e.g. in an inactivated form.

[0079] In one embodiment, the modified gelatinized potato starch composition is dried so that it has a water content of less than 20 wt.%, preferably less than 15 wt.%, more preferably less than 10 wt.%. Food ingredient or food product

[0080] Another aspect of the invention relates to a food ingredient or food product comprising the potato starch composition according to the invention.

[0081] In one embodiment, the food product is selected from the group consisting of a cheese product, e.g. a normal cheese and a cheese analogue, gummy candies, a spread, a dressing, a pudding, vanilla cream, a sauce, a mayonnaise, an ice cream, a yogurt, a dessert, a dough, a baking cream, a baked good, a coffee creamer, a baby food, a soup and a noodle. Use of modified potato starch

[0082] Another aspect of the invention relates to the use of a modified potato starch according to the invention as a gelling agent and / or texturizer, such as for gelling cheese, such as a normal cheese or a cheese analogue, for gelling gummy candies or as a gelatin substitute.

[0083] It should be noted that embodiments and features described in the context of one aspect of the present invention also apply to the other aspects of the invention.

[0084] All patent and non-patent references cited in the present application are hereby incorporated in their entirety by reference.

[0085] The invention will now be described in more detail in the following non-limiting examples. Examples Example 1 - Materials and Methods Enzyme production and purification:

[0086] The α-amylase-encoding genes were codon-optimized for expression in E. coli, synthesized, and cloned in-frame in pET28a-TEV using the restriction sites Ndel and BamHl (Genscript, Piscataway, NJ, USA) with an N-terminal His tag. E. coli BL21(DE3) was used as the expression host and routinely inoculated in LB broth containing 50 µg ml- 1 Kanamycin increases at 37°C until an OD 600 of 0.4–0.6 was achieved. The cultures were placed on ice for 10 minutes before expression was induced by the addition of 0.5 mM IPTG and 3 mM lactose, followed by overnight expression ( ~Cells were incubated for 18 hours at 16°C. Cells were harvested by centrifugation (5500 g), sonified on ice (Qsonica ultrasound system, Qsonica, Newtown, CT, USA), and supernatants were obtained by centrifugation (19,500 g, 4°C, 20 min). His-tagged proteins were purified by loading sterile, filtered supernatants onto a 5 ml Histrap™ FF crude column (GE Healthcare, UK), washed with binding buffer (20 mM HEPES, pH 7.4, 500 mM NaCl, 20 mM imidazole), and eluted using a 50–500 mM imidazole gradient. The purity of the proteins was verified by SDS-PAGE analysis and the protein concentrations were determined spectrophotometrically at 280 nm using the theoretical extinction coefficients of 103,550 (µm), 144,090 (Po) and 203,120 M. -1 cm -1 (Ac) determined.

[0087] The expressed enzymes can be identified as described below. enzyme species Family Genebank deposit number Ac Anaerocolumna cellulosilytica GH13_42 WP_197978574.1 Po Petrotoga mobilis GH13_5 WP_012208959.1 To uncultured marine bacterium GH13_37 ADK21254.1

[0088] The expressed enzymes exhibit the following SEQ ID NOs: name SEQ ID NO : Ac 1 Po 2 To 3 Modification of gelatinized starch with α-amylases:

[0089] 10% (dry matter) native potato starch (KMC - Superior Potato Starch) was gelatinized at 95°C with 5.6 mM CaCl₂ using a Vorwerk Thermomix. The solution was portioned into bottles with blue caps, cooled to 50°C, and maintained at this temperature using a water bath with a magnetic stirrer. The respective enzyme was added to 100 g of the solution and allowed to react with the starch for 5–60 minutes at 50°C. After the reaction, the enzyme was inactivated by adding NaOCl at room temperature and subsequently NaHSO₃ (both at a final concentration of 4 mM). For functional analyses, the solution was portioned into plastic beakers or RVA aluminum containers (see below) and stored at 5°C. For structural analyses, the solution was diluted to an enzymatically modified gelatinized starch concentration of 1% (dry matter) and stored at room temperature. High-performance size exclusion chromatography (HPSEC) for determining molecular weight:

[0090] The 1% enzymatically modified gelatinized potato starch solutions were diluted to 0.2% in 100 mM NaOAc buffer, pH 6. The starch solutions were then filtered through 0.45 µm nylon filters and analyzed by HPSEC using a Vanquish quaternary pump and a Vanquish autosampler connected to an ERC RefractoMax 520 refractive index detector (ThermoFisher Scientific, Waltham, MA, USA). The column used was a Shodex OHpak SB-806M HQ (300 x 8 mm) equipped with a Shodex OHpak SB-G guard column (50 x 6 mm, Showa Denko KK, Tokyo, Japan). The samples (100 µl) were analyzed using an isocratic eluent of 100 mM NaOAc, pH 6, for 35 minutes at a flow rate of 0.5 ml / min.Pullulan standards in the range of 180–1,450,000 Da (PSS Polymer Standards Service GmbH, Mainz, Germany) were used to calculate the molecular weight based on an extrapolation of a third-degree polynomial expression. Differences in the response factor for starch molecules of different sizes were not taken into account when integrating the signals for molecular weight intervals (Example 4). High-performance anion exchange chromatography with amperometric detection (HPAEC-PAD) for determining chain lengths:

[0091] The 1% enzymatically gelatinized starch solutions were diluted to 0.2% in 50 mM NaOAc, pH 4. The starch molecules were branched overnight at 45°C with isoamylase (0.1 U / mg starch, Megazyme, Bray, Ireland), and the isoamylase was subsequently inactivated by heating to 95°C for 10 minutes. The starch solutions were then filtered through 0.45 µm nylon filters and analyzed by HPAEC-PAD using a Dionex ICS-6000 SP system (Thermo Fisher Scientific, Waltham, MA, USA). A CarboPac PA200 column (3 x 250 / 50 mm, Thermo Fisher Scientific, Waltham, MA, USA) was used as the column and guard column.The samples (5 µl) were analyzed using a constant flow rate of 0.5 mL / min and an isocratic eluent of 0.2 M NaOH (0–48 min) combined with a gradient of NaOAc for elution of the starch chains (0–15 min: 50–225 mM, 15–35 min: 225–350 mM, 35–45 min: 350–375 mM, 45–47 min: 375–500 mM, 47–48 min: 500 mM), followed by a wash step with 1 M NaOAc and equilibration to 50 mM NaOAc. Glucose and maltooligosaccharides of DP2-8 were used as standards for determining the chain lengths of the analyzed samples. The signals corresponding to DP1-90 were integrated individually, and for each chain length a “% total” was calculated, indicating the percentage share of each chain length compared to the sum of all chains with DP1-90.

[0092] To better illustrate the changes made to the gelatinized starch by the respective enzymes, the change in the chain length distribution “Δ% total” was also calculated by comparing it to the “1% total” for native potato starch (Superior Potato Starch, KMC). Both definitions are used in Example 5. The calculated percentages are based solely on the integration of the signals, without taking into account the variation in the response factor between the different chain lengths. Bicinchoninic acid (BCA) assay for the determination of reducing ends:

[0093] The amount of reducing ends in enzymatically modified gelatinized potato starch was determined using the BCA assay with D-glucose as a standard. The 1% starch solutions were diluted 7.5-fold in BCA solution (1:1:1 solution A, solution B, and deionized water). The samples were then incubated at 75°C for 30 minutes, cooled to room temperature, and the absorbance at 560 nm was measured against a blank sample containing deionized water. Standard curves were generated using dilutions from a 400 µM D-glucose solution. The reducing ends were calculated as µmol / g starch.

[0094] Solution A: 2.4 g Na2CO3, 1.1 g NaHCO3 and 87.4 mg BCA disodium salt hydrate in 45 g deionized water

[0095] Solution B: 56.2 mg CuSO4 pentahydrate and 56.8 mg L-serine in 45 g deionized water. Iodine assay for determining amylose content:

[0096] The amylose content of enzymatically modified gelatinized potato starches was determined using the I₂ / Ki assay. 175 µl of the 1% starch solution were placed in a 5 mL Eppendorf tube. 225 µl of 1 M NaOH were added, and the mixture was allowed to stand for 10 minutes. 2200 µl of deionized water were added. 100 µl of the mixture was transferred to a 2 mL Eppendorf tube. For a blank sample, this was replaced with 0.09 M NaOH. Subsequently, 1840 µl of deionized water, 20 µl of AcOH, and 40 µl of an iodine / Ki solution (0.2% / 2% in deionized water) were added. After 20–30 minutes, the absorbance at 550 and 620 nm was measured against a blank sample.

[0097] The standard curves were prepared using dilutions of a 0.5 mg / ml amylose solution and a 2 mg / ml amylopectin solution, both in 0.09 M NaOH.

[0098] The amylose content can be determined as described here: Amylose%=100%∗aap620−aap550∗R(aam550−aap550)∗R−(aam620−aap620)

[0099] Here, a is the slope, determined using standard curves for amylose (am) and amylopectin (ap) at 550 and 620 nm. R is the ratio of the measured absorptions Abs620 / Abs550. Rheology

[0100] The 10% enzymatically modified gelatinized solutions (30 g), stored at 5°C in plastic beakers (Ø 60 mm), were analyzed using a TA HR-20 instrument. A frequency sweep followed by an amplitude sweep was performed using a 40.0 mm cross-nested parallel-plate geometry. Both analyses were carried out at 25°C with a spacing of 1000 µm. The frequency sweep was performed at 0.5% strain from 0.1 to 20 Hz, and the amplitude sweep was performed at 1 Hz and from 0.01 to 1000% strain. The values ​​given in the examples are all derived from the amplitude sweep and were measured within the linear viscoelastic range (LVR), which is defined as the range with a linear relationship between the complex modulus and the vibrational strain. The “end of the LVR” is defined as the vibration strain (%) at which the complex modulus has decreased by 5% relative to the LVR. Texture analysis

[0101] The 10% enzymatically modified gelatin solutions (28 g), stored at 5°C in RVA aluminum cans (Ø 37 mm, Perten, Stockholm, Sweden), were analyzed using a TA.XT Plus Texture Analyzer (Stable Micro Systems, Godalming, Surrey, UK) equipped with a 5 kg load cell. A cylindrical probe (P / 0.5R) was used to penetrate 5 mm into the starch solution (trigger force 5 g, 0.5 mm / s). The hardness was determined as the force (g) after 1.5 seconds. Syneresis

[0102] The 10% enzymatically modified gelatinized solutions (25 g), stored at 5°C in plastic beakers (Ø 35 mm), were transferred to an aluminum ring (Ø 60 mm) placed on filter paper (Ø 270 mm, technical grade, 15 µm). If necessary, the sample was compressed to fit the surface of the aluminum ring. Any free water from the sample was allowed to pass through the filter paper. After 10 minutes, a line was drawn to indicate the distance to which the water had passed through. The distance from the aluminum ring was measured at 8 points evenly distributed around the circumference of the ring, and a value for syneresis (in mm) was calculated as the mean of these measurements. Fig. 1). Example 2 - Gelling properties Objective of the analysis

[0103] Determination of the gelling properties of enzymatically modified gelatinized potato starches obtained using the enzymes Um, Ac and Po. Materials and methods

[0104] The modified potato starches were produced according to Example 1.

[0105] Details are given in Table 1 and Table 2 using the enzymes Um, Ac, and Po, as well as two commercially available α-amylases from IFF (New York City, New York, USA), Spezyme FRED and GC007, derived from Bacillus licheniformis and Bacillus stearothermophilus, respectively. Both commercially available enzymes are commonly used in the enzymatic depolymerization of starch. Table 1: enzyme Enzyme stock solution (per 100 g of starch solution) Response time (min) Strength #1 To 37 µl 5 Strength #2 To 37 µl 40 Strength #3 Ac 14 µl 5 Strength #4 Ac 14 µl 60 Strength #5 Po 888 µl 40 Strength #6 SpezymeFRED 1,36 µl 20 Strength #7 GC007 0,74 µl 40 Table 2: enzyme Concentration, enzyme stock solution To 34766 nM Ac 107808 nM Po 8328 nm Spezyme FRED 18904 LU / g GC007 41200 LAU / g

[0106] The units are specified according to the manufacturer's instructions for commercially available enzymes. Results

[0107] As shown in Table 3, starch #1 (Um) and starch #6 (specyme FRED) have comparable amylose content and reducing ends, indicating the same degree of hydrolysis. However, a solution of starch #1 (stored for 2 days at 5°C) exhibits a much higher complex modulus and much higher hardness than starch #6, which is due to a greater tendency for starch #1, modified with the enzyme Um, to form a gel structure. This is also evident from the figures in Fig. 2 stands out. Table 3 enzyme Amylose (%) Reducing ends (µmol / g starch) G* (Well) hardness (g) Strength #1 To 19,7 24,8 1585 60,4 Strength #6 SpezymeFRED 19,2 29,6 286 15,5

[0108] A comparison of the rheological measurements of starch #3 (Ac) and starch #5 (Po) with starch #7 (GC007) (see Table 4) reveals that starch #3 (Ac) in particular, but also starch #5 (Po), despite the similarity of the complex modulus to that of starch #7 (GC007), exhibit a much higher end of the LVR than starch #7 (GC007), which suggests greater stability against shaking. Table 4: enzyme Amylose (%) Reducing ends (µmol / g starch) G* (Well) End of the LVR (%) Strength #3 Ac 14,1 15 45,5 10 Strength #4 Ac 9,5 70 36,3 1,1 Strength #5 Po 16,1 98 41,2 1,6 Strength #7 GC007 16,1 63 43,7 0,77 conclusion

[0109] Example 2 shows that gelatinized potato starches modified with the enzymes Um, Ac and Po have significantly better gelling properties and higher stability compared to potato starches modified with commercially available α-amylases. Example 3 - Syneresis: Goal of the analysis

[0110] Determination of the syneresis of enzymatically modified gelatinized potato starches obtained using the enzymes Um, Ac and Po after storage at 5°C. Materials and methods

[0111] Enzymatically modified gelatinized potato starches were produced according to Example 1.

[0112] Details are given in Tables 5 and 6 using the enzymes (Ac, Po, and Um), GC007 (IFF), or Spezyme Endurase (IFF) shown therein. The starch solutions were stored at 5°C for 2, 7, and 14 days prior to syneresis measurements, as per Example 1. Table 5: enzyme Enzyme stock solution (µl / 100 g starch solution) Response time (min) Strength #8 Ac 14 10 Strength #9 Ac 14 30 Strength #4 Ac 14 60 Strength #10 Po 888 5 Strength #11 Po 888 20 Strength #12 Po 888 60 Strength #13 GC007 0,74 10 Strength #14 GC007 0,74 30 Strength #15 GC007 0,74 60 Strength #1 To 37 5 Strength #16 To 37 20 Strength #17 To 37 60 Strength #18 Specyme Endurase 49 10 Strength #19 Specyme Endurase 49 30 Strength #20 Specyme Endurase 49 60 Table 6: Concentration, enzyme stock solution Ac 107808 nM Po 8328 nm GC007 41200 LAU / g To 34766 nM Specyme endurase 58742 LPAU / g Results

[0113] Table 7 compares the syneresis of starch solutions from Table 5 after 7 and 14 days at 5°C. It is evident that starch solutions based on gelatinized potato starches modified with Ac, Po, and Um exhibit a significantly lower tendency to release water after storage at 5°C than solutions based on starches modified with commercially available enzymes. Table 7: enzyme Storage at 5°C (days) Syneresis (mm) Strength #8 Ac 7 0,5 Strength #9 Ac 7 1,1 Strength #4 Ac 7 4,1 Strength #10 Po 7 0 Strength #11 Po 7 1,0 Strength #12 Po 7 4,8 Strength #13 GC007 7 3,0 Strength #14 GC007 7 17,1 Strength #15 GC007 7 15,5 Strength #1 To 14 5,3 Strength #16 To 14 4,4 Strength #17 To 14 6,2 Strength #18 Specyme Endurase 14 2,1 Strength #19 Specyme Endurase 14 25,5 Strength #20 Specyme Endurase 14 33,9

[0114] The starch solutions #4, #12 and #14 are listed in Table 8 and Fig. 3 further comparisons. Despite similar values ​​for G' and G'' after 7-day storage, starch solution #14 (GC007) has a significantly stronger tendency to release water compared to starch solution #4 (Ac) and starch solution #12 (Po). Table 8: enzyme Store at 5°C (Take) Syneresis (mm) G' (Well) G'' (Well) Strength #4 Ac 7 4,1 71,2 22,8 Strength #12 Po 7 4,8 70,7 19,0 Strength #14 GC007 7 17,1 78,1 14,2

[0115] Furthermore, the starch solutions #16 and #19 are shown in Table 9 and Fig.4 compared. Despite similar values ​​for G* and tan(δ) after 14-day storage, starch solution #19 (specyme endurase) has a significantly stronger tendency to release water compared to starch solution #16 (Um). Table 9: enzyme Store at 5°C (Take) Syneresis (mm) G* (Well) tan (d) Strength #16 To 14 4,4 1389 0,16 Strength #19 Specyme Endurase 14 25,5 1303 0,09 conclusion

[0116] Starch solutions based on gelatinized potato starches modified with the enzymes Ac, Po and Um have a significantly lower tendency to release water (lower syneresis) after storage than potato starches modified with commercially available α-amylases. Example 4 - Molecular weight distribution of starches Objective of the analysis

[0117] Evaluation of the molecular weight distribution of enzymatically modified gelatinized starches. Materials and methods

[0118] Enzymatically modified gelatinized starches were produced according to Examples 1 and 2. Results

[0119] Fig. Figure 5 shows the molecular weight distribution of starch #1 (Um), starch #2 (Um), and starch #6 (specyme FRED) from Example 2. It is clearly evident that starch #6 has a much more centrally centered molecular weight distribution compared to starch #1, which, despite the comparable degree of degradation as shown in Example 2, exhibits a larger fraction of large molecules. A starch that was degraded to a greater extent than starch #1, but with the same enzyme (starch #2), still exhibits a much larger fraction of large molecules as well as a larger fraction of small molecules compared to starch #6. This demonstrates the different pattern of starch hydrolysis for these enzymes, which could explain the differences in functional properties.

[0120] Fig.Figure 6 shows the molecular weight distribution of starch #3 (Ac), starch #4 (Ac), starch #5 (Po), and starch #7 (GC007) from Example 2. Again, starch #7, produced with one of the commercially available α-amylases, exhibits a larger fraction of medium-sized starch molecules. On the other hand, starch #3, starch #4, and starch #5 all exhibit a much larger fraction of large molecules, even though starch #4 and starch #5 have a greater number of reducing ends (Table 4), again demonstrating a different reaction pattern for the enzymes shown here.

[0121] To account for these differences in molecular weight distributions, as in Fig. 5 and Fig.To further quantify the results shown in Figure 6, the ratio between the medium molecular weight starch fraction and the low and high molecular weight starch fraction was calculated for each enzymatically modified starch. The ratio R is given as follows: R = ∫ medium molecular weight power fraction ∫ low molecular weight power fraction + ∫ high molecular weight power fraction Low molecular weight starch fraction: Mw = 5.0 * 10 2 - 3.3 * 10 4 Since the mean molecular weight of the starch fraction is: Mw = 3.4 * 10 4 - 8.9 *10 5 Since the starch fraction has a high molecular weight: Mw = 9.0 * 10 5 - 4.0 *10 7 There.

[0122] The R-values ​​calculated individually for each enzymatically modified starch are given in Table 10b and summarized for each enzyme in Table 10a. Table 10a: enzyme reaction time (min) R Ac 5-60 0,27-0,60 Po 5-60 0,33-0,50 To 5-60 0,31-0,39 Spezyme FRED 5-60 0,43-1,81 Specyme endurase 5-60 0,49-1,53 GC007 5-60 0,40-1,43 Table 10b: enzyme reaction time (min) R Ac 5 0,27 Ac 10 0,60 Ac 40 0,40 Ac 60 0,46 Po 5 0,33 Po 10 0,37 Po 20 0,41 Po 40 0,45 Po 60 0,50 To 5 0,33 To 10 0,31 To 20 0,32 To 40 0,35 To 60 0,39 Spezyme FRED 5 0,43 Spezyme FRED 10 0,68 Spezyme FRED 20 1,12 Spezyme FRED 40 1,81 Spezyme FRED 60 1,68 Specyme endurase 5 0,49 Specyme endurase 10 0,62 Specyme endurase 20 1,17 Specyme endurase 40 1,53 Specyme endurase 60 1,35 GC007 5 0,40 GC007 10 0,55 GC007 20 0,80 GC007 40 1,25 GC007 60 1,43 conclusion

[0123] The enzymatically modified gelatinized potato starches obtained with the enzymes Ac, Po and Um exhibit different molecular weight distribution profiles compared to the tested commercially available amylases (Spezyme FRED, Spezyme Endurase and GC007), which suggests Fig. 5 and Fig. 6, as can be seen from the low R-values ​​in Tables 10a-10b. Furthermore, Um exhibits non-overlapping R-values ​​that are lower than those of commercially available enzymes.

[0124] Without being bound to any particular theory, it is assumed that this difference in the molecular weight distribution profiles contributes to the differences in functional properties described in Examples 2 and 3. Example 5 - Chain length distribution of potato starches. Objective of the analysis

[0125] Evaluation of the chain length distribution of enzymatically modified gelatinized potato starches. Materials and methods

[0126] Enzymatically modified gelatinized potato starches were produced according to Examples 1 and 2. Results

[0127] The different reaction patterns of the enzymes presented here (Um, Ac, Po) compared to the commercially available enzymes tested can also be seen in the chain length distribution of the modified starches. The differences are particularly pronounced in the region of starch chains consisting of 1-10 glucose units (DP1-10), as shown in Fig. 7-11 are shown and quantified in various ways in Tables 11-13. Change in chain length distribution “Δ% total”:

[0128] When examining the change in the chain length distribution “Δ% total” compared to a native strength (explained in Example 1) of strengths #1 and #2 (Um) ( Fig. 7) and strength #6 (Specyme FRED) ( Fig. 8) Starches #1 and #2 (Um) show a greater increase at DP1-5 than at DP6-10 after enzymatic modification than starch #6 (specyme FRED). These differences are quantified in Table 11b and summarized for each enzyme in Table 11a. Table 11a: enzyme reaction time (min) Δ%Total(DP1−5)Δ%Total(DP6−10) Ac 5-60 0,45-0,61 Po 5-60 0,17-0,45 To 5-60 1,83-2,15 Spezyme FRED 5-60 0,39-0,74 Specyme endurase 5-60 0,41-0,63 GC007 5-60 0,28-0,52 Table 11b: enzyme reaction time (min) Δ%Total(DP1−5)Δ%Total(DP6−10) Ac 5 0,45 Ac 10 0,55 Ac 20 0,50 Ac 40 0,58 Ac 60 0,61 Po 5 0,17 Po 10 0,21 Po 20 0,28 Po 40 0,38 Po 60 0,45 To 5 2,15 To 10 1,90 To 20 1,83 To 40 2,01 To 60 1,97 Spezyme FRED 5 0,39 Spezyme FRED 10 0,43 Spezyme FRED 20 0,53 Spezyme FRED 40 0,58 Spezyme FRED 60 0,74 Specyme endurase 5 0,43 Specyme endurase 10 0,41 Specyme endurase 20 0,44 Specyme endurase 40 0,52 Specyme endurase 60 0,63 GC007 5 0,30 GC007 10 0,28 GC007 20 0,39 GC007 40 0,45 GC007 60 0,52

[0129] Tables 11a and 11b show that the starches modified with the enzyme Um do not have overlapping values ​​for the ratio between DP1-5 and DP6-10, demonstrating a unique chain length profile for the starches modified with Um. Chain length distribution “% total”:

[0130] The presence of a lower amount of starch chains with DP6-10 in starches modified with μ-10 compared to other enzymatically modified starches can also be quantified by the sum of the "% total" values ​​for the starch chains with a degree of polymerization of 6-10 glucose units. These values ​​are given in Table 12b and summarized for each enzyme in Table 12a. Table 12a: enzyme reaction time (min) % Total (DP6 - 10) Ac 5-60 9,4-13,9 Po 5-60 10,5-15,7 To 5-60 9,7-11,3 Spezyme FRED 5-60 11,7-17,3 Specyme endurase 5-60 11,6-17,9 GC007 5-60 13,1-21,2 Table 12b: enzyme reaction time (min) % Total (DP6 - 10) Ac 5 9,4 Ac 10 9,9 Ac 20 10,9 Ac 40 12,5 Ac 60 13,9 Po 5 10,5 Po 10 11,2 Po 20 12,7 Po 40 14,6 Po 60 15,7 To 5 9,7 To 10 10,1 To 20 10,6 To 40 10,9 To 60 11,3 Spezyme FRED 5 11,7 Spezyme FRED 10 12,5 Spezyme FRED 20 13,9 Spezyme FRED 40 16,3 Spezyme FRED 60 17,3 Specyme endurase 5 11,6 Specyme endurase 10 12,4 Specyme endurase 20 14,4 Specyme endurase 40 16,6 Specyme endurase 60 17,9 GC007 5 13,1 GC007 10 14,9 GC007 20 15,9 GC007 40 19,0 GC007 60 21,2

[0131] The values ​​above indicate that the starches modified with Um do not overlap with those modified with the commercially available enzymes examined with regard to the amount of DP6-DP10. Furthermore, the starches modified with Ac and Po generally also show lower values ​​than the commercially modified starches, although there is some overlap in values ​​when long incubation times of Ac and Po are compared with short incubation times of the commercially available enzymes.

[0132] The change in the chain length distribution of strengths #3 (Ac), #4 (Ac) and #5 (Po) is in Fig. Figures 9-11 illustrate this. A closer look at the change in chain length of chains consisting of 1-10 glucose units reveals again the magnitude of the increase in DP1 and D6 in starch #3 (Ac), #4 (Ac) and #5 (Po) compared to starch #7 (GC007), which exhibits the greatest increase in DP7.

[0133] To quantify these differences, the ratio between the amount of DP1 and DP6 compared to DP2-5 and DP7-10 was again calculated for the enzymatically modified starches (Table 13b) and summarized for each enzyme (Table 13a). Table 13a: enzyme reaction time (min) %Total(DP1+DP6)%Total(DP2−5+DP7−10) Ac 5-60 0,36-0,58 Po 5-60 0,42-0,54 To 5-60 0,23-0,28 Spezyme FRED 5-60 0,22-0,29 Specyme endurase 5-60 0,20-0,29 GC007 5-60 0,23-0,29 Table 13b: enzyme reaction time (min) %Total(DP1+DP6)%Total(DP2−5+DP7−10) Ac 5 0,36 Ac 10 0,39 Ac 20 0,45 Ac 40 0,52 Ac 60 0,58 Po 5 0,39 Po 10 0,42 Po 20 0,45 Po 40 0,50 Po 60 0,54 To 5 0,28 To 10 0,26 To 20 0,25 To 40 0,23 To 60 0,23 Spezyme FRED 5 0,29 Spezyme FRED 10 0,28 Spezyme FRED 20 0,26 Spezyme FRED 40 0,23 Spezyme FRED 60 0,22 Specyme endurase 5 0,29 Specyme endurase 10 0,28 Specyme endurase 20 0,24 Specyme endurase 40 0,22 Specyme endurase 60 0,20 GC007 5 0,29 GC007 10 0,28 GC007 20 0,26 GC007 40 0,24 GC007 60 0,23

[0134] These calculated values ​​show that the starches modified by Ac and Po exhibit high, non-overlapping values ​​with other modified starches, demonstrating the unique reaction patterns for these enzymes. conclusion

[0135] The enzymatically modified gelatinized potato starches obtained with the enzymes Ac, Po and Um exhibit different chain length distribution profiles compared to the commercially available amylases (Spezyme FRED, Spezyme Endurase and GC007).

[0136] Um stands out significantly when it comes to the amount of starch chains with DP1-5 compared to DP6-10, by showing non-overlapping high values ​​for the relationship between DP1-5 and DP6-10 (see Tables 11a and 11b), but also when it comes to the amount of DP6-DP10 alone, by showing non-overlapping low values ​​(see Tables 12a and 12b).

[0137] Ac and Po stand out significantly in terms of the relationship between DP1 and DP6 compared to DP 2-5 and DP7-10, exhibiting non-overlapping high values ​​(see Table 13a and Table 13b).

[0138] Without being bound to any theory, it is assumed that this difference in the chain length distribution profiles contributes to the differences in functional properties described in Examples 2 and 3. Points of the invention

[0139] 1. A method for producing a modified potato starch composition, the method comprising a) Providing a gelatinized potato starch; b) Providing one or more alpha amylases selected from the group consisting of enzyme species Genebank deposit number To uncultured marine bacterium ADK21254.1 Ac Anaerocolumna cellulosilytica WP_197978574.1 Po Petrotoga mobilis WP_012208959.1 c) Incubating the gelatinized potato starch from step a) with one or more alpha-amylases from step b), for example for a period of 5 - 180 minutes at a temperature in the range of 40 - 70°C; d) if necessary, inactivating one or more alpha-amylases; and e) Providing a modified potato starch composition.

[0140] 2. Method according to point 1, wherein in step a) the gelatinized potato starch is selected from the group consisting of a gelatinized native potato starch, a gelatinized chemically modified potato starch, a gelatinized enzymatically modified potato starch and a gelatinized physically modified potato starch or combinations thereof.

[0141] 3. Procedure according to point 1 or 2, wherein in step a) - the gelatinized potato starch is selected from the group consisting of waxy potato starch, potato starch with an amylose content of the starch of over 20% by weight, for example over 30% by weight; and / or - the gelatinized chemically modified potato starch is selected from the group consisting of acid-hydrolyzed starch, succinate starch such as octenylsuccinate starch, hydroxypropyl starch, oxidized starch, cross-linked starch such as diphosphate starch or adipic acid cross-linked starch, acetylated starch and phosphorylated starch; and / or - the gelatinized, physically modified potato starch is selected from the group consisting of heat-treated starch, such as dry-heated starch, heat-moisture-treated starch and tempered starch, pressure-treated starch, extruded starch, drum-dried starch, spray-dried starch, radiation-treated starch, such as ultrasonically treated starch and microwave-treated starch; and / or - the gelatinized enzymatically modified potato starch is selected from the group consisting of starches modified by glycoside hydrolases such as other alpha-amylases, beta-amylases, glucoamylases, maltogenic alpha-amylases and branching enzymes (e.g. pullulanase and isoamylase), glycosyltransferases such as 4-alpha-glucanotransferases, branching enzymes and cyclodextrin glucanotransferases.

[0142] 4. A method according to any of the preceding points, wherein the gelatinized potato starch from step a) is produced using a method selected from the group consisting of heating to at least 60°C, preferably at least 80°C, more preferably at least 95°C, such as heating in the presence or absence of cations, such as CaCl2.

[0143] 5. Method according to any of the preceding points, wherein the one or more alpha amylases are defined by - an amino acid sequence selected from the group consisting of SEQ ID NO: 1 (Ac), SEQ ID NO:2 (Po) and SEQ ID NO:3 (Um), or - an amino acid sequence with at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 1 (Ac), SEQ ID NO:2 (Po) and SEQ ID NO:3 (Um), such as at least 95% sequence identity, such as 98% sequence identity or such as 99% sequence identity.

[0144] 6. Method according to one of the preceding points, wherein the temperature during step c) is in the range of 20 - 90°C, e.g. 20 - 40°C, e.g. 40 - 60°C or e.g. 70 - 90°C.

[0145] 7. Procedure according to one of the above points, wherein step c) is carried out over a period of time in the range of 5 minutes to 120 minutes, e.g. 5 minutes to 60 minutes.

[0146] 8. A method according to any of the preceding points, wherein the one or more alpha amylases are inactivated by a method selected from the group consisting of: • NaOCl treatment, preferably followed by a reduction using NaHSO3; • Heat inactivation, e.g. heating to at least 90°C; • low pH value, e.g. below pH 5, e.g. below pH 3; and • high pH value, e.g. above pH 10, e.g. above pH 12.

[0147] 9. Method according to any of the preceding points, wherein the starch in the provided potato starch composition comprises: I) - a ratio of the degree of glucose polymerization of % Total (DP1+DP6)% Total (DP2−5+DP7−10) above 0.32, e.g. above 0.34; and / or - a ratio of the degree of glucose polymerization of % Total (DP1+DP6)% Total (DP2−5+DP7−10) in the range 0.33 - 0.7, e.g. in the range 0.35 - 0.7; preferably in the range 0.35 - 0.6; and / or II) - a glucose polymerization degree % of total (DP6 - 10) below 11.4; and / or - a glucose polymerization degree % of total (DP6 - 10) in the range 9 - 11.3, e.g. 9.2 - 11, e.g. 9.5 - 10; and / or III) - Δ% Total(DP1−5)Δ% Total(DP6−10)>1 preferably above 1.5; and / or - Δ%Total(DP1−5)Δ%Total(DP6−10) in the range 1 - 2.5, preferably in the range 1.5 - 2.3, more preferably in the range 1.7 - 2.2; where Δ% of total is calculated as a comparison with a corresponding gelatinized potato starch before enzymatic modification; and / or IV) - a molecular weight distribution (R) below 0.6; e.g. in the range 0.2 - 0.59, preferably in the range 0.25 - 0.4; and more preferably in the range 0.25 - 0.35; where the molecular weight distribution (R) is defined as R = ∫ medium molecular weight power fraction ∫ low molecular weight power fraction + ∫ high molecular weight power fraction Low molecular weight starch fraction: Mw = 5.0 * 10 2 - 3.3 * 10 4 Since the mean molecular weight of the starch fraction is: Mw = 3.4 * 10 4 - 8.9 *10 5 Since the starch fraction has a high molecular weight: Mw = 9.0 * 10 5 -4.0 *10 7 There.

[0148] 10. Procedure according to one of the preceding points, wherein step e) comprises the following steps: - e1) Drying the starch; and - e2) Providing a dried modified potato starch composition.

[0149] 11. Method according to point 10, wherein the drying step e1) is carried out by a method selected from the group consisting of spray drying, flash drying, drum drying and freeze drying.

[0150] 12. Modified potato starch composition obtained or available by a process according to any of the preceding points.

[0151] 13. Use of an alpha amylase selected from the group consisting of enzyme species Genebank deposit number To uncultured marine bacterium ADK21254.1 Ac Anaerocolumna cellulosilytica WP_197978574.1 Po Petrotoga mobilis WP_012208959.1 for the modification of gelatinized potato starch.

[0152] 14. Use of an alpha amylase as defined in point 13, to produce a modified starch with improved gelling and / or texturizing properties and / or reduced syneresis.

[0153] 15. Use as per point 14, wherein the improved gelling properties include increased hardness and / or a higher complex modulus.

[0154] 16. Having a modified gelatinized potato starch composition - a ratio of the degree of glucose polymerization of % Total (DP1+DP6)% Total (DP2−5+DP7−10) above 0.32, e.g. above 0.34; and / or - a ratio of the degree of glucose polymerization of % Total (DP1+DP6)% Total (DP2−5+DP7−10) in the range of 0.33 - 0.7, e.g. in the range of 0.35 - 0.7; preferably in the range of 0.35 - 0.6. 17. Having a modified gelatinized potato starch composition or having a modified gelatinized potato starch composition as per point 16 - a glucose polymerization degree % of total (DP6 - 10) below 11.4; and / or - a glucose polymerization degree % of total (DP6 - 10) in the range 9 - 11.3, e.g. 9.2 - 11, e.g. 9.5 - 10. 18. Having a modified gelatinized potato starch composition or having a modified gelatinized potato starch composition as defined in point 16 or 17 - Δ% Total(DP1−5)Δ% Total(DP6−10)>1, preferably above 1.5; and / or - Δ% Total(DP1−5)Δ% Total(DP6−10) in the range 1 - 2.5, preferably in the range 1.5 - 2.3, stronger preferably in the range 1.7 - 2.2; where Δ% total is calculated as a comparison with a corresponding gelatinized potato starch before enzymatic modification.

[0155] 19. Modified gelatinized potato starch composition or modified gelatinized potato starch composition according to any of points 16 to 18, having a degree of molecular weight distribution (R) below 0.6, e.g. in the range 0.2 - 0.59, preferably in the range 0.25 - 0.4; and more preferably in the range 0.25 - 0.35; where the molecular weight distribution (R) is defined as R = ∫ medium molecular weight power fraction ∫ low molecular weight power fraction + ∫ high molecular weight power fraction Low molecular weight starch fraction: Mw = 5.0 * 10 2 - 3.3 * 10 4 Since the mean molecular weight of the starch fraction is: Mw = 3.4 * 10 4 - 8.9 *10 5 Since the starch fraction has a high molecular weight: Mw = 9.0 * 10 5 - 4.0 *10 7 There.

[0156] 20. Modified gelatinized potato starch composition according to any of items 16 to 19, comprising one or more enzymes selected from: enzyme species Genebank deposit number To uncultured marine bacterium ADK21254.1 Ac Anaerocolumna cellulosilytica WP_197978574.1 Po Petrotoga mobilis WP_012208959.1 preferably wherein one or more enzymes are inactivated.

[0157] 21. Modified gelatinized potato starch composition according to any one of points 16 to 20, which is dried so that it has a water content of less than 20 wt.%, preferably less than 15 wt.%, more preferably less than 10 wt.%.

[0158] 22. Food ingredient or food product comprising the potato starch composition according to any of points 16 to 21.

[0159] 23. Food product as defined in point 22, wherein the food product is selected from the group consisting of a cheese product, e.g. a normal cheese and cheese analogues, gummy candies, a spread, a dressing, a pudding, vanilla cream, a sauce, a mayonnaise, an ice cream, a yogurt, a dessert, a dough, a baking cream, a baked good, a coffee creamer, a baby food, a soup and a noodle.

[0160] 24. Use of a modified potato starch composition according to point 12 or one of points 16 to 21 as a gelling agent and / or texturizer, for example for gelling cheese, e.g. normal cheese or cheese analogues, for gelling gummy candies, as a gelatin substitute or as a fat substitute.

[0161] SEQ ID NO: 1-3 are shown without wt signal peptides. Signal peptides or purification tags can be added, for example, at the N- or C-terminus. SEQ ID NO: 4 is the wt signal peptide for Ac, SEQ ID NO: 5 is the wt signal peptide for Po, and SEQ ID NO: 6 is an example His tag. QUOTES INCLUDED IN THE DESCRIPTION

[0000] This list of documents cited by the applicant was automatically generated and is included solely for the reader's convenience. The list is not part of the German patent or utility model application. The DPMA accepts no liability for any errors or omissions. Cited non-patent literature

[0000] Zinck et al. (Molecules 2023, 28, 2947

[0005] Liu et al. (Mar Biotechnol (2012) 14:253-260

[0006] http: / / www.paralign.org

[0041]

Claims

[1] Modified potato starch composition obtained or available by a process comprising the steps of: f) Providing gelatinized potato starch; g) Providing one or more alpha amylases selected from the group consisting of enzyme species Genebank deposit number To uncultured marine bacterium ADK21254.1 Ac Anaerocolumna cellulosilytica WP_197978574.1 Po Petrotoga mobilis WP_012208959.1 h) Incubating the gelatinized potato starch from step a) with one or more alpha-amylases from step b), for example for a period of 5 - 180 minutes at a temperature in the range of 40 - 70°C; i) optionally inactivating one or more alpha-amylases; and j) Providing a modified potato starch composition. [2] Modified potato starch composition obtained or available by the process of claim 1, wherein the starch in the provided potato starch composition comprises: I) - a ratio of the degree of glucose polymerization of % Total (DP1+DP6)% Total (DP2−5+DP7−10) above 0.32, e.g. above 0.34; and / or - a ratio of the degree of glucose polymerization of % Total (DP1+DP6)% Total (DP2−5+DP7−10) in the range 0.33 - 0.7, e.g. in the range 0.35 - 0.7; preferably in the range 0.35 - 0.6; and / or II) - a glucose polymerization degree % of total (DP6 - 10) below 11.4; and / or - a glucose polymerization degree % of total (DP6 - 10) in the range 9 - 11.3, e.g. 9.2 - 11, e.g. 9.5 - 10; and / or III) - Δ% Total(DP1−5)Δ% Total(DP6−10)>1, preferably above 1.5; and / or - Δ% Total(DP1−5)Δ% Total(DP6−10) in the range 1 - 2.5, preferably in the range 1.5 - 2.3, more preferably in the range 1.7 - 2.2; where Δ% of total is calculated as a comparison with a corresponding gelatinized potato starch before enzymatic modification; and / or IV) - a molecular weight distribution (R) below 0.6; e.g. in the range 0.2 - 0.59, preferably in the range 0.25 - 0.4; and more preferably in the range 0.25 - 0.35; where the molecular weight distribution (R) is defined as R = ∫ medium molecular weight power fraction ∫ low molecular weight power fraction + ∫ high molecular weight power fraction Low molecular weight starch fraction: Mw = 5.0 * 10 2 - 3.3 * 10 4 Since the mean molecular weight of the starch fraction is: Mw = 3.4 * 10 4 - 8.9 *10 5 Since the starch fraction has a high molecular weight: Mw = 9.0 * 10 5 - 4.0 *10 7 There. [3] Having a modified potato starch composition - a ratio of the degree of glucose polymerization of % Total (DP1+DP6)% Total (DP2−5+DP7−10) above 0.32, e.g. above 0.34; and / or - a ratio of the degree of glucose polymerization of % Total (DP1+DP6)% Total (DP2−5+DP7−10) in the range of 0.33 - 0.7, e.g. in the range of 0.35 - 0.7; preferably in the range of 0.35 - 0.

6. [4] Having a modified gelatinized potato starch composition or a modified gelatinized potato starch composition according to claim 3 - a glucose polymerization degree % of total (DP6 - 10) below 11.4; and / or - a glucose polymerization degree % of total (DP6 - 10) in the range 9 - 11.3, e.g. 9.2 - 11, e.g. 9.5 - 10. [5] Having a modified gelatinized potato starch composition or a modified gelatinized potato starch composition according to claim 3 or 4 - Δ% Total(DP1−5)Δ% Total(DP6−10)>1, preferably above 1.5; and / or - Δ% Total(DP1−5)Δ% Total(DP6−10) in the range 1 - 2.5, preferably in the range 1.5 - 2.3, more preferably in the range 1.7 - 2.2; where Δ% total is calculated as a comparison with a corresponding gelatinized potato starch before enzymatic modification. [6] Modified gelatinized potato starch composition or modified gelatinized potato starch composition according to any one of claims 3 to 5, having a degree of molecular weight distribution (R) below 0.6, e.g. in the range 0.2 - 0.59, preferably in the range 0.25 - 0.4; and more preferably in the range 0.25 - 0.35; where the molecular weight distribution (R) is defined as R = ∫ medium molecular weight power fraction ∫ low molecular weight power fraction + ∫ high molecular weight power fraction Low molecular weight starch fraction: Mw = 5.0 * 10 2 - 3.3 * 10 4Since the mean molecular weight of the starch fraction is: Mw = 3.4 * 10 4 - 8.9 *10 5 Since the starch fraction has a high molecular weight: Mw = 9.0 * 10 5 - 4.0 *10 7 There. [7] Modified gelatinized potato starch composition according to any one of claims 3 to 6, comprising one or more enzymes selected from: enzyme species Genebank deposit number To uncultured marine bacterium ADK21254.1 Ac Anaerocolumna cellulosilytica WP_197978574.1 Po Petrotoga mobilis WP_012208959.1 preferably wherein one or more enzymes are inactivated. [8] Modified potato starch composition according to any one of claims 3 to 7, which is dried so that it has a water content of less than 20 wt% water, preferably less than 15 wt%, more preferably less than 10 wt% water. [9] Food ingredient or food product comprising the potato starch composition according to any one of claims 1 to 8. [10] Food product according to claim 9, wherein the food product is selected from the group consisting of a cheese product, e.g. a normal cheese and cheese analogues, gummy candies, a spread, a dressing, a pudding, vanilla cream, a sauce, a mayonnaise, an ice cream, a yogurt, a dessert, a dough, a baking cream, a baked good, a coffee creamer, a baby food, a soup and a noodle.

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