Method for producing a low methoxylation amidated pectin

Description

35 METHOD FOR PRODUCING A LOW METHOXYLATION AMIDATED PECTIN CROSS-REFERENCE TO RELATED DEPOSIT REQUESTS

[001] This application claims the benefit of European patent application no. 23167014.2, filed on April 6, 2023, which is incorporated herein by reference in its entirety. FIELD OF THE INVENTION

[002] The present invention relates to an amidated pectin and a method of manufacturing amidated pectin. In particular, the present invention relates to an amidated pectin formed from a wet precipitated pectin of high methoxylation. BACKGROUND OF THE INVENTION

[003] Pectin is found in nature, in the cell walls of plants. Although pectin can be extracted from a large number of plants or fruits, the main industrial or conventional sources include citrus peel and apple pomace. The main use of pectin is as a gelling agent, thickening agent, viscosifying agent and / or as a stabilizer in food. As a result, pectin is commonly used in jams, jellies, acidic dairy drinks and dessert bases as a food additive. Pectin can also be used as dietary fiber and as an effective food emulsifier. Pectin also has potential uses in other fields, such as in medicine, as a vehicle for controlled drugs or bioactive release, and in many other non-food applications.

[004] Pectin is a polysaccharide comprising a chain of galacturonic acid units linked by α1,4 glycosidic bonds. The galacturonic acid chain is partially esterified as methyl esters. Although the galacturonic acid chain is the backbone of pectin, other chemicals, such as acetyl and methyl ester groups, Petition 870250088676, dated 09 / 30 / 2025, page 11 / 52 / 35 can be incorporated into the chain. These groups may be important for distinguishing between different types of pectin. An analysis of the properties and characteristics of pectin can be found in D. Gawkowska et al. “Structure-Related Gelling of Pectins and Linking with Other Natural Compounds: A Review” Polymers (Basel), July 2018; 10(7): 762, the disclosure of which is incorporated here by reference.

[005] Pectin can be divided into two categories by its degree of methylation, which is the percentage of carboxyl groups that have undergone esterification with methanol. The degree of methylation is of great importance for the use of pectins as gelling agents and, depending on the degree of methylation, pectin is classified as a high methoxyl pectin (HM pectin) or a low methoxyl pectin (LM pectin). If more than 50 percent of the carboxyl groups are methylated, then the pectin is an HM pectin, and if less than that degree of methylation has occurred, then the pectin is an LM pectin.

[006] HM pectin is the most commonly used pectin and can be divided into two categories: fast-setting HM pectin and slow-setting HM pectin. Fast-setting pectin will set more quickly and at a higher temperature than slow-setting pectin and is therefore preferable in the production of food and / or beverage products that typically require a certain mouthfeel or the suspension of various particulate materials such as whole or chopped fruit, nuts and the like. Slow-setting pectin is preferred for food and / or beverage products that do not require the presence of particulate matter in suspension. HM pectins can form gels with sugar and acid, forming what is known in the art as low water activity gels or sugar-acid pectin gels. Slow-setting pectin can take 1 to 2 days to turn into a gel.These gels have a three-dimensional network of pectin molecules in which the... Petition 870250088676, dated 09 / 30 / 2025, p. 12 / 52 / 35 solvent (usually water), optionally with sugar and acid cosolutes, are immobilized, resulting in a system that can resist deformation.

[007] LM pectins differ from HM pectins, where pectins LM pectins require only the presence of a divalent metal ion to initiate the reaction that causes pectin to transform into a gel. The most commonly used divalent metal ion is calcium. In general, the gel-forming capacity of LM pectins increases with a decreasing degree of methylation.

[008] Recently, LM pectins have been amidated to form amidated LM pectin. Amidated LM pectin is a modified form of LM pectin in which some of the galacturonic acid units are converted by ammonia into carboxylic amides. In traditional methods of producing amidated LM pectin, HM pectin must first be extracted from its natural source. The HM pectin is converted into LM pectin, which undergoes amidation to form amidated LM pectin. The conversion of HM pectin into amidated LM pectin follows the reaction scheme: RCOOCH3 + NH4OH => RCONH2 + CH3OH +H2O

[009] Traditional methods of producing amidated LM pectin from natural pectin in citrus fruits are presented below. Traditional Method 1: Traditional method of producing an amidated LM pectin from natural pectin derived from citrus fruits, wherein the traditional method includes a dry amidation step at a temperature of 10 to 14 °C.

[0010] Traditional method 1 refers to an example of producing amidated LM pectin using traditional methods. In traditional method 1, HM pectin is first extracted from citrus fruit peel and then dried. The extracted and dried HM pectin then undergoes amidation at a temperature of 10 to 14 °C to form amidated LM pectin. Petition 870250088676, dated 09 / 30 / 2025, page 13 / 52 / 35

[0011] Figure 1 represents a flow diagram for the traditional method 1. Step 1: Extraction of HM pectin from citrus fruit peel

[0012] First, citrus peel is mixed in hot acidic water to extract HM pectin in aqueous form from the citrus peel. The temperature and pH of the hot acidic water is 68 to 72 °C, the pH is 1.50 to 1.70, and the residence time is 8 to 12 hours.

[0013] HM pectin in aqueous form is clarified using centrifugation, filtration or other conventional separation technologies known in the art to obtain clarified HM pectin in aqueous form.

[0014] The clarified HM pectin in aqueous form is then concentrated according to any technique known in the art and an appropriate amount of an alcohol is added to form an alcohol / water mixture. The amount of alcohol added depends on the amount of alcohol required to cause the clarified HM extract to precipitate out of its aqueous form as a precipitate containing the HM pectin.

[0015] The precipitate containing HM pectin is separated from the alcohol / water mixture by mechanical separation, and any mechanical separation known in the art may be used. The HM pectin obtained from the separation is then washed three times with 60% alcohol (by weight, of the total weight of the alcohol) to form washed HM pectin.

[0016] Washed HM pectin is dried and ground until a homogeneous particle size distribution is achieved to form dry HM pectin. Generally, dry HM pectin has a moisture content of 10% by weight of the total weight of dry HM pectin. Dry HM pectin can be mixed with sugar to form standardized HM pectin. Step 2: Amidation of dry HM pectin

[0017] Dry HM pectin is placed in a suspension that Petition 870250088676, dated 09 / 30 / 2025, page 14 / 52 / 35, comprises alcohol and ammonia. Alcohol is present at 60 to 70% by weight of the total weight of the suspension. Ammonia is present at 2.8 to 3.5% by weight of the total weight of the suspension. The suspension is maintained at a temperature of 10 to 14 °C. The dry HM pectin remains in suspension for 5 to 8 hours. After 5 to 8 hours, the dry HM pectin undergoes amidation to form amidated LM pectin.

[0018] Amidated LM pectin is filtered from the suspension by any filtration technique known in the field. The alcohol and ammonia pass to a recovery step.

[0019] Amidated LM pectin is washed with 60% alcohol, by weight, of the total weight of the alcohol to form washed amidated LM pectin.

[0020] The pH of the washed amidated LM pectin is adjusted until the pH is between 4.0 and 4.5. The pH is adjusted with an acid, such as nitric acid. The result is an amidated LM pectin with an adjusted pH.

[0021] The pH-adjusted amidated LM pectin is washed with 60% alcohol, by weight, of the total weight of the alcohol three times to form a second washed amidated LM pectin.

[0022] The second washed amidated LM pectin undergoes a drying process to form a dry amidated LM pectin with a moisture content of 10% by weight of the total weight of the dry amidated LM pectin. Any drying processes known in the art may be used.

[0023] The dried amidated LM pectin is then ready for quality analysis and further modification. Further modifications may include standardization with sugar to make a commercial product.

[0024] Typically, the alcohol used in traditional method 1 is isopropyl alcohol. Traditional Method 2: A traditional method for producing an amidated LM pectin from natural pectin derived from citrus fruits, wherein the traditional method includes a calcium removal step and a dry amidation step at a temperature of 12 to 14 °C. Petition 870250088676, dated 09 / 30 / 2025, page 15 / 52 / 35

[0025] Traditional method 2 refers to an example of producing amidated LM pectin using traditional methods. In traditional method 2, HM pectin is first extracted from citrus fruit peel and then dried. The extracted and dried HM pectin then undergoes amidation at a temperature of 12 to 14 °C to form amidated LM pectin.

[0026] Figure 2 represents a flow diagram for the traditional method 2. Step 1: Extraction of HM pectin from a citrus fruit peel.

[0027] First, citrus peel is mixed in hot acidic water to extract HM pectin in aqueous form from the citrus peel. The temperature of the hot acidic water is 70 to 80 °C, the pH is 1.90 to 2.10, and the residence time is 3 to 3.5 hours.

[0028] HM pectin in aqueous form is clarified using centrifugation, filtration or other conventional separation technologies known in the art to obtain clarified HM pectin in aqueous form.

[0029] The calcium present in the clarified HM pectin in aqueous form is removed using ion exchange with a cationic resin to form a decalcified HM pectin in aqueous form.

[0030] Decalcified HM pectin in aqueous form is concentrated, according to any technique known in the art, to form concentrated HM pectin in aqueous form.

[0031] The degree of esterification (DE) of concentrated aqueous HM pectin is modified by adding an acidic solution to the concentrated aqueous HM pectin. Typically, the acid used is nitric acid, sulfuric acid, and / or hydrochloric acid. The degree of esterification (DE) is modified at a temperature of 30 to 35 °C, at an acidity of 0.80 to 1.0% by mass, and with a residence time of 72 to 96 hours. The resulting product is an HM pectin with a modified DE in the form Petition 870250088676, dated 09 / 30 / 2025, p. 16 / 52 / 35 aqueous, and HM pectin with modified DE in aqueous form has a degree of esterification (DE) of 54 to 58%.

[0032] An appropriate amount of alcohol is added to the modified DE HM pectin in aqueous form to form an alcohol / water mixture. The amount of alcohol added depends on the amount of alcohol required to cause the modified DE HM pectin to precipitate from its aqueous form as a precipitate containing the HM pectin.

[0033] The precipitate containing HM pectin is separated from the alcohol / water mixture by mechanical separation, and any mechanical separation technique known in the art can be used.

[0034] The precipitate containing HM pectin is then washed three times with 60% alcohol, by weight, of the total weight of the alcohol to form a washed HM pectin.

[0035] Washed HM pectin is dried and ground until a homogeneous particle size distribution is achieved to form dry HM pectin. Generally, dry HM pectin has a moisture content of 10% by weight of the total weight of dry HM pectin. Dry HM pectin can be mixed with sugar to form standardized HM pectin. Step 2: Amidation of dry HM pectin

[0036] Dried HM pectin is placed in a suspension comprising alcohol and ammonia. The alcohol is present at 60 to 70% by weight of the total weight of the suspension. The ammonia is present at 2.8 to 3.5% by weight of the total weight of the suspension. The suspension is maintained at a temperature of 12 to 14 °C. The dried HM pectin remains in the suspension for 5 to 8 hours. After 5 to 8 hours, the dried HM pectin undergoes amidation to form amidated LM pectin.

[0037] Amidated LM pectin is removed by filtration from the suspension. Alcohol and ammonia are then passed to a recovery step.

[0038] Amidated LM pectin is washed with 60% alcohol, by weight, of the total weight of the alcohol to form washed amidated LM pectin. Petition 870250088676, dated 09 / 30 / 2025, page 17 / 52 / 35

[0039] The pH of the washed amidated LM pectin is adjusted until the pH is between 4.0 and 4.5. The pH is adjusted with an acid, such as nitric acid. The result is an amidated LM pectin with an adjusted pH.

[0040] The pH-adjusted amidated LM pectin is washed with 60% alcohol, by weight, of the total weight of the alcohol three times to form a second washed amidated LM pectin.

[0041] The second washed amidated LM pectin undergoes a drying process to form a dry amidated LM pectin with a moisture content of 10% by weight of the total weight of the dry amidated LM pectin. Any drying processes known in the art may be used.

[0042] Amidated LM pectin is then ready for quality analysis and further modification. Further modifications may include standardization with sugar to make a commercial product.

[0043] Typically, the alcohol used in traditional method 2 is isopropyl alcohol. Traditional Method 3: This is a traditional method for producing amidated LM pectin from natural pectin derived from citrus fruits. The traditional method includes a calcium removal step and a dry amidation step at a temperature of 7 to 8 °C.

[0044] Traditional method 3 refers to an example of producing amidated LM pectin using traditional methods. In traditional method 3, HM pectin is first extracted from citrus fruit peel and dried. The extracted and dried HM pectin then undergoes amidation at a temperature of 7 to 8 °C to form amidated LM pectin.

[0045] Figure 2 represents a flow diagram for the traditional method 3. Step 1: Extraction of HM pectin from a citrus fruit peel.

[0046] First, citrus fruit peel is mixed in hot acidic water to extract HM pectin in aqueous form from the peel. Petition 870250088676, dated 09 / 30 / 2025, page 18 / 52 / 35 citrus fruits. The temperature of the acidic hot water is 70 to 80 °C, the pH is 1.90 to 2.10 and the residence time is 3 to 3.5 hours.

[0047] HM pectin in aqueous form is clarified using centrifugation, filtration or other conventional separation technologies known in the art to obtain clarified HM pectin in aqueous form.

[0048] The calcium present in the clarified HM pectin in aqueous form is removed using ion exchange with a cationic resin to form a decalcified HM pectin in aqueous form.

[0049] Decalcified HM pectin in aqueous form is concentrated, according to any technique known in the art, to form concentrated HM pectin in aqueous form.

[0050] The degree of esterification (DE) of concentrated aqueous HM pectin is modified by adding an acidic solution to the concentrated aqueous HM pectin. Typically, the acid used is nitric acid, sulfuric acid, and / or hydrochloric acid. The degree of esterification (DE) is modified at a temperature of 30 to 35 °C, at an acidity of 0.80 to 1.0% by mass, and with a residence time of 72 to 96 hours. The resulting product is aqueous HM pectin with modified DE and has a degree of esterification (DE) of 54 to 58%.

[0051] An appropriate amount of alcohol is added to the modified DE HM pectin in aqueous form to form an alcohol / water mixture. The amount of alcohol added depends on the amount of alcohol required to cause the HM pectin to precipitate from its aqueous form as a precipitate containing the HM pectin.

[0052] The precipitate containing HM pectin is separated from the alcohol / water mixture by mechanical separation, and any mechanical separation technique known in the art may be used.

[0053] The precipitate containing HM pectin is then washed three times Petition 870250088676, dated 09 / 30 / 2025, page 19 / 52 / 35 times with 60% alcohol, by weight, of the total weight of the alcohol to form a washed HM pectin.

[0054] Washed HM pectin is dried and ground until a homogeneous particle size distribution is achieved to form dry HM pectin. Generally, dry HM pectin has a moisture content of 10% by weight of the total weight of dry HM pectin. Dry HM pectin can be mixed with sugar to form standardized HM pectin. Step 2: Amidation of dry HM pectin

[0055] Dried HM pectin is placed in a suspension comprising alcohol and ammonia. The alcohol is present at 60 to 70% by weight of the total weight of the suspension. The ammonia is present at 4.0 to 5.0% by weight of the total weight of the suspension. The suspension is maintained at a temperature of 7 to 8 °C. The dried HM pectin remains in the suspension for 3 to 5 hours. After 3 to 5 hours, the dried HM pectin undergoes amidation to form amidated LM pectin.

[0056] The amidated LM pectin is then removed by filtration from the suspension. The alcohol and ammonia are then taken to a recovery step.

[0057] Amidated LM pectin is washed with 60% alcohol, by weight, of the total weight of the alcohol to form washed amidated LM pectin.

[0058] The pH of the washed amidated LM pectin is adjusted until the pH is between 4.0 and 4.5. The pH is adjusted with an acid, such as nitric acid. The result is an amidated LM pectin with an adjusted pH.

[0059] The pH-adjusted amidated LM pectin is washed with 60% alcohol, by weight, of the total weight of the alcohol three times to form a second washed amidated LM pectin.

[0060] The second washed amidated LM pectin undergoes a drying process to form a dry amidated LM pectin with a moisture content of 10% by weight of the total weight of the dry amidated LM pectin. Any drying processes known in the art may be used.

[0061] Dried amidated LM pectin is then ready for analysis of Petition 870250088676, dated 09 / 30 / 2025, page 20 / 52 / 35 quality and additional modification. Additional modifications may include standardization with sugar to make a commercial product.

[0062] Typically, the alcohol used in traditional method 3 is isopropyl alcohol.

[0063] Other methods of forming amidated LM pectin are set out in documents US2480710A and WO98 / 58968A1.

[0064] The product of well-known methods may undergo glycosidic degradation and aggregation during amidation, which leads to the amidated pectin having an altered conformation due to the degradation caused during production.

[0065] There is therefore a need for an improved amidated LM pectin, and the improved amidated LM pectin has the same or similar properties to LM pectin.

[0066] There is therefore a need for an improved method of producing an amidated LM pectin, and the method does not cause HM pectin to undergo glycosidic degradation and aggregation during the production of amidated LM pectin. SUMMARY OF THE INVENTION

[0067] The present invention relates to an amidated pectin and a method of manufacturing amidated pectin. In particular, the present invention relates to an amidated pectin formed from a wet precipitated high-methoxyl pectin.

[0068] The representative features of the present invention are defined in the following clauses, which are independent or may be combined, in any combination, with one or more of the features disclosed in the text and / or figures of the description.

[0069] The present invention is described in the following clauses: 1. A method for producing a low-methoxyl amidated pectin, the method comprising the following step: Petition 870250088676, dated 09 / 30 / 2025, p. 21 / 52 / 35 to add a precipitated wet pectin of high methoxylation to a suspension comprising one or more alcohols and ammonia to form an amidated pectin of low methoxylation.

[0070] 2. The method of clause 1, with the low methoxylated amidated pectin having a degree of methylation below 50%.

[0071] 3. The method of clause 1 or clause 2, wherein the wet precipitated pectin of high methoxylation has a degree of methylation above 50%.

[0072] 4. The method of any of clauses 1 to 3, wherein the wet precipitated pectin of high methoxylation has an intrinsic viscosity of 500 to 650 mL / g, or 510 to 640 mL / g, or 525 to 615 mL / g.

[0073] 5. The method of any of clauses 1 to 4, wherein the low methoxylated amidated pectin has an intrinsic viscosity of 450 to 650 mL / g, or 475 to 625 mL / g, or 488 to 600 mL / g.

[0074] 6. The method of any of clauses 1 to 5, wherein the precipitated wet pectin of high methoxylation is in suspension with one or more alcohols and ammonia at a temperature of 1 to 12 °C, or 2 to 10 °C, or 3 to 8 °C, or 4 to 6 °C.

[0075] 7. The method of any of clauses 1 to 6, wherein the precipitated wet pectin of high methoxylation is in suspension with one or more alcohols and ammonia for a reaction time of 1 to 8 hours, or 2 to 7 hours, or 2.5 to 6.5 hours, or 3 to 6 hours.

[0076] 8. The method of any of clauses 1 to 7, wherein the wet precipitated pectin of high methoxylation has a dry matter content of 20 to 80% by weight, or 30 to 70% by weight, or 40 to 60% by weight, or 45 to 55% by weight, or 50% by weight, of the total weight of the wet precipitated pectin of high methoxylation.

[0077] 9. The method of any of clauses 1 to 8, wherein ammonia is present in the suspension from 1 to 10% by weight, or from 2 to 8% by weight. Petition 870250088676, dated 09 / 30 / 2025, page 22 / 52 / 35 weight, or from 3 to 7%, by weight, or from 4 to 6%, by weight, of the total weight of the suspension.

[0078] 10. The method of any of clauses 1 to 9, wherein one or more alcohols are present in the suspension in 50 to 80% by weight, or 55 to 75% by weight, or 60 to 70% by weight of the total weight of the suspension.

[0079] 11. The method of any of clauses 1 to 10, wherein one or more alcohols are any of tert-amyl alcohol, benzyl alcohol, 1,4-butanediol, 1,2,4-butanetriol, butanol, 1-butanol, 2-butanol, tert-butyl alcohol, diethylene glycol, ethanol, ethylene glycol, 2-ethylhexanol, furfuryl alcohol, glycerol, isobutanol, isopropyl alcohol, methanol, 2-(2-methoxyethoxyl)ethanol, 2-methyl-1-butanol, 2-methyl-1-pentanol, 3-methyl-2-butanol, neopentyl alcohol, 2-pentanol, 1,3-propanediol, propan-1-ol, propylene glycol, propylene glycol methyl ether, isopropanol or any combination thereof.

[0080] 12. The method of any of clauses 1 to 11, which additionally includes the step of: filtering the low-methoxyl amidated pectin from the suspension to form a filtrate comprising one or more alcohols and ammonia and a residue comprising the low-methoxyl amidated pectin, wherein the filtering step of the low-methoxyl amidated pectin from the suspension interrupts the reaction between the high-methoxyl pectin, one or more alcohols and ammonia.

[0081] 13. The method of clause 12, which additionally comprises the step of: Neutralize the residue comprising low methoxylated amidated pectin with acidulated alcohol until the residue comprising low methoxylated amidated pectin has a pH of 3.0 to 5.0, or 4.0 to 5.0, or 4.0 to 4.5.

[0082] 14. The method of clause 13, wherein the acidified alcohol Petition 870250088676, dated 09 / 30 / 2025, page 23 / 52 / 35 includes a mineral acid; optionally, the mineral acid being nitric acid; Optionally, the acidulated alcohol further comprises any of tert-amyl alcohol, benzyl alcohol, 1,4-butanediol, 1,2,4-butanetriol, butanol, 1-butanol, 2-butanol, tert-butyl alcohol, diethylene glycol, ethanol, ethylene glycol, 2-ethylhexanol, furfuryl alcohol, glycerol, isobutanol, isopropyl alcohol, methanol, 2-(2-methoxyethoxyl)ethanol, 2-methyl-1-butanol, 2-methyl-1-pentanol, 3-methyl-2-butanol, neopentyl alcohol, 2-pentanol, 1,3-propanediol, propan-1-ol, propylene glycol, propylene glycol methyl ether, isopropanol, or any combination thereof.

[0083] 15. The method of any of clauses 12 to 14, further comprising removing or extracting water and / or alcohol from the residue comprising low methoxylated amidated pectin.

[0084] 16. The method of clause 15, whereby the removal or extraction of water and / or alcohol from the residue comprising low methoxylated amidated pectin is carried out by pressing.

[0085] 17. The method of clause 16, wherein the pressing step is carried out until the low methoxylated amidated pectin has a dry matter content of 30 to 70% by weight, or 40 to 60% by weight, or 45 to 55% by weight, or 50% by weight, of the total weight of the low methoxylated amidated pectin.

[0086] 18. The method of any of clauses 1 to 17, which additionally comprises a drying step, whereby water and / or alcohol is removed from the low methoxylated amidated pectin.

[0087] 19. The method of clause 18, wherein the drying step is carried out until the low methoxylated amidated pectin has a moisture content of 1 to 30% by weight, or 2 to 20% by weight, or 7.5 to 15.0% by weight, or 9 to 11% by weight, or 10% by weight, of the total weight of the low methoxylated amidated pectin. Petition 870250088676, dated 09 / 30 / 2025, p. 24 / 52 / 35

[0088] 20. The method of any of clauses 1 to 19, which additionally includes the step of: Grind low-methoxyl amidated pectin; and / or mix low-methoxyl amidated pectin with sugar.

[0089] 21. The method of any of clauses 1 to 20, wherein the precipitated wet pectin of high methoxylation is produced by the following method: Mix a watery citrus peel in hot acidic water until a highly methoxylated pectin is extracted from the citrus peel; centrifuge, filter and / or concentrate highly methoxylated pectin; Add an acidic solution to high-methoxyl pectin to modify the degree of esterification (DE) of the high-methoxyl pectin; Add alcohol to high methoxyl pectin until a precipitated high methoxyl pectin is formed; and separate the precipitated high methoxyl pectin from the alcohol to form a wet precipitated high methoxyl pectin.

[0090] 22. The method of clause 21, wherein the citrus peel is derived from lemons, tangerines, grapefruits, sweet oranges, pomelos, clementines, limes, kumquats, bitter oranges, blood oranges, satsumas, yuzus, tangelos or a combination thereof.

[0091] 23. The method of clause 21 or clause 22, wherein the step of mixing aqueous citrus fruit peel in hot acidic water, until high methoxyl pectin is extracted from the citrus fruits, is carried out at a temperature of 55 to 100 °C, or 60 to 90 °C, or 65 to 85 °C, or 70 to 80 °C.

[0092] 24. The method of any of clauses 21 to 23, the step being to mix watery citrus fruit peel in hot water. Petition 870250088676, dated 09 / 30 / 2025, p. 25 / 52 / 35 acidic, until high methoxylation pectin is extracted from citrus fruits, is conducted at a pH of 1.5 to 2.5, or 1.75 to 2.25, or 1.9 to 2.10.

[0093] 25. The method of any of clauses 21 to 24, wherein the step of mixing aqueous citrus fruit peel in hot acidic water, until the highly methoxylated pectin is extracted from the citrus fruit, is carried out for 2 to 4.5 hours, or 2.5 to 4 hours, or 3 to 3.5 hours.

[0094] 26. The method of any of clauses 21 to 25, wherein acidic hot water comprises any of nitric acid, phosphoric acid, sulfuric acid, citric acid, malic acid, succinic acid, adipic acid, gluconic acid, tartaric acid, fumaric acid or a combination thereof.

[0095] 27. The method of any of clauses 21 to 26, which additionally comprises the step of removing calcium present in high methoxyl pectin by ion exchange.

[0096] 28. The method of any of clauses 21 to 27, wherein the high methoxylation pectin is concentrated until the HM pectin is present in water at a concentration of 15 to 40 g / L, preferably 20 to 37 g / L, more preferably 25 to 35 g / L, most preferably 27 to 30 g / L.

[0097] 29. The method of any of clauses 21 to 28, wherein the step of adding an acidic solution to high methoxyl pectin to modify the degree of esterification (DE) of the high methoxyl pectin is carried out to a degree of esterification of 40 to 75%, or 45 to 70%, or 50 to 65%, or 54 to 58%.

[0098] 30. The method of any of clauses 21 to 29, wherein the step of adding an acidic solution to high methoxyl pectin to modify the degree of esterification (DE) of the high methoxyl pectin is carried out at a temperature of 20 to 45 °C, or 25 to 40 °C, or 30 to 35 °C. W. Petition 870250088676, dated 09 / 30 / 2025, p. 26 / 52 / 35

[0099] 31. The method of any of clauses 21 to 30, wherein the step of adding an acid solution to high methoxyl pectin to modify the degree of esterification (DE) of the high methoxyl pectin is carried out until the acid content is 0.5 to 1.5% by weight, or 0.6 to 1.25% by weight, or 0.7 to 1.1% by weight, of the total weight of the combined acid and high methoxyl pectin.

[00100] 32. The method of any of clauses 21 to 31, wherein the step of adding an acidic solution to high methoxyl pectin to modify the degree of esterification (DE) of the high methoxyl pectin is carried out for 50 to 110 hours, or 60 to 105 hours, or 70 to 100 hours, or 72 to 96 hours.

[00101] 33. The method of any of clauses 21 to 32, wherein the step of adding an acidic solution to high methoxyl pectin to modify the degree of esterification (DE) of the high methoxyl pectin is carried out with any of the following acids: nitric acid, sulfuric acid, hydrochloric acid, phosphoric acid, citric acid, malic acid, succinic acid, adipic acid, gluconic acid, tartaric acid, fumaric acid or a combination thereof; optionally, and the acid is present in water.

[00102] 34. The method of any of clauses 21 to 33, which further comprises removing or extracting water from the wet precipitated pectin of high methoxylation.

[00103] 35. The method of clause 34, wherein the removal or extraction of water from the wet precipitated pectin of high methoxylation is carried out by pressing, optionally, wherein the wet precipitated pectin of high methoxylation is pressed until the wet precipitated pectin of high methoxylation has a dry substance of 30 to 70% by weight, or 40 to 60% by weight, or 50% by weight, of the total weight of the wet precipitated pectin of high methoxylation. Petition 870250088676, dated 09 / 30 / 2025, p. 27 / 52 / 35

[00104] 36. Method for forming a dairy product, optionally for use in milk, condensed milk, dried milk, dairy drinks, cream, butter, yogurt, cheese, custard, cottage cheese, cream cheese, curd, frozen dairy products such as frozen custard, frozen yogurt and ice cream, gelato, evaporated milk, evaporated milk, cream, curdled milk, whey, whey protein or whipped cream, the method comprising the steps of incorporating the product of any of clauses 1 to 35 into a dairy product.

[00105] 37. A low methoxylated amidated pectin obtained, or obtainable, by the method of any of clauses 1 to 35.

[00106] 38. Low methoxylated amidated pectin of clause 37 for use in dairy products, optionally for use in milk, condensed milk, dried milk, dairy drinks, cream, butter, yogurt, cheese, custard, cottage cheese, cream cheese, curd, frozen dairy products such as frozen custard, frozen yogurt and ice cream, gelato, milk powder, evaporated milk, curdled milk, whey, whey protein or whipped cream. BRIEF DESCRIPTION OF THE DRAWINGS

[00107] Figure 1 is a flow diagram of a traditional method for producing amidated LM pectin from dry HM pectin.

[00108] Figure 2 is a flow diagram of a traditional method for producing amidated LM pectin from dry HM pectin.

[00109] Figure 3 is a flow diagram of the method according to the present invention for producing an amidated LM pectin from a wet precipitated HM pectin.

[00110] Figure 4 is a graph showing the differences in intrinsic viscosity between HM pectin and the same pectin after amidation for amidated LM pectins formed through the traditional dry amidation process and amidated LM pectin formed through the method of the present invention. DETAILED DESCRIPTION Petition 870250088676, dated 09 / 30 / 2025, p. 28 / 52 / 35

[00111] The embodiments of the invention are described below with reference to the accompanying drawings. The accompanying drawings illustrate various embodiments of systems, methods, and embodiments of various other aspects of the disclosure. Anyone skilled in the art will appreciate that the element boundaries illustrated (e.g., boxes, groups of boxes, or other shapes) in the figures represent an example of the boundaries. It may be that, in some examples, an element can be designed as multiple elements or that multiple elements can be designed as one element. In some examples, an element shown as an internal component of one element can be implemented as an external component in another, and vice versa. Furthermore, the elements may not be drawn to scale. Non-limiting and non-exhaustive descriptions are given with reference to the following drawings. The components in the figures are not necessarily to scale, the emphasis being placed on illustrative principles.

[00112] The embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings, in which like numerals represent like elements throughout the various figures and in which exemplary embodiments are shown. The embodiments of the claims may, however, be embodied in many different forms and should not be interpreted as being limited to the embodiments set forth herein.

[00113] The words “comprise”, “have”, “contain”, and “include”, and other forms thereof, are intended to be equivalent in terms and are open-ended by the fact that an item, or items, following any of these words is not intended to be an exhaustive listing of such item, or items, or is intended to be limited only to the item or items listed. It should also be noted that, as used herein and in the appended claims, the singular forms “a”, “an”, “the”, and “the” include plural references unless the context clearly indicates otherwise. Although any systems and methods Petition 870250088676, dated 09 / 30 / 2025, p. 29 / 52 / 35 similar or equivalent to those described in this document may be used in the practice or testing of embodiments of the present disclosure, the preferred systems and methods are now described.

[00114] Some of the terms used to describe the present invention are presented below: "Amidation" refers to the modification of a pectin to include at least one amide group (-NH2).

[00115] “Degree of methylation” refers to the percentage of carboxyl groups esterified with methanol. If more than fifty percent of the carboxyl groups are methylated, the pectin is called high methoxyl pectin (HM), and if less than that degree of methylation has occurred, then the pectin is called low methoxyl pectin (LM).

[00116] “High methoxylation pectin” or “HM pectin” refers to pectin extracted from citrus fruit peels, wherein a degree of the original carboxyl groups present in the pectin have been subjected to esterification with methanol. High methoxylation pectins have a degree of methylation above 50%.

[00117] “Low methoxylation pectin” or “LM pectin” refers to pectin extracted from citrus fruit peels, where a degree of the original carboxyl groups present in the pectin have been esterified with methanol. Low methoxylation pectins have a degree of methylation below 50%.

[00118] “Low methoxylated amidated pectin” or “amidated LM pectin” or “LMA pectin” refers to a modified form of LM pectin in which some of the galacturonic acid residues are converted by ammonia into amide groups.

[00119] “Blender” or “mixture” refers to a substance containing two or more different components.

[00120] “Wet precipitate” refers to a substance that has a Petition 870250088676, dated 09 / 30 / 2025, p. 30 / 52 / 35 moisture content of 20 to 80% by weight, or 30 to 70% by weight, or 40 to 60% by weight, or 45 to 55% by weight, or 50% by weight, of the total weight of the substance. Preferably, the moisture content is 50% by weight of the total weight of the substance. The substance may be HM pectin with a moisture content of 50% by weight of the total weight of the HM pectin. A method for producing an amidated LM pectin from natural citrus fruit pectin, and the method includes a wet precipitate amidation step.

[00121] According to a first aspect of the present disclosure, a method is provided for producing amidated LM pectin from a natural pectin derived from citrus fruits. In particular, the method requires amidation of a wet precipitated HM pectin.

[00122] Advantageously, the amidation of a wet precipitated HM pectin results in an amidated LM pectin that has not undergone the same degree of glycosidic degradation during amidation as traditional pectin amidation, which requires the HM pectin amidation to occur while the HM pectin is dry. Advantageously, the resulting amidated LM pectin has similar physical properties, such as intrinsic viscosity, as the HM pectin before amidation.

[00123] To obtain amidated LM pectin, wet precipitated HM pectin is first extracted from a natural pectin source, such as citrus fruits. The wet precipitated HM pectin is then subjected to amidation to form amidated LM pectin.

[00124] Figure 3 represents a flow diagram for a method according to the first aspect of the present disclosure. Step 1: Extraction of HM pectin from a natural pectin source.

[00125] HM pectin is extracted from a natural source of pectin. Natural sources of pectin can be fruits such as apples, pears, plums, guavas, quince, currants, and citrus fruits. Examples of citrus fruits include, but Petition 870250088676, dated 09 / 30 / 2025, p. 31 / 52 / 35 are not limited to lemons, tangerines, grapefruits, sweet oranges, pomelos, clementines, limes, kumquats, bitter oranges, blood oranges, satsumas, yuzus, tangelos, or a combination thereof. Preferably, HM pectin is extracted from citrus peel.

[00126] HM pectin is extracted from a natural source by placing the natural source in hot acidic water.

[00127] Optionally, the temperature of the acidic hot water is 55 to 100 °C, preferably 60 to 90 °C, more preferably 65 to 85 °C, and most preferably 70 to 80 °C.

[00128] Optionally, the pH of acidic hot water is 1.5 to 2.5, preferably 1.75 to 2.25, and with a maximum preference of 1.9 to 2.10.

[00129] Optionally, the pH of acidic hot water is controlled by adding an acid to the hot water. The acid can be any one or more of nitric acid, phosphoric acid, sulfuric acid, citric acid, malic acid, succinic acid, adipic acid, gluconic acid, tartaric acid, fumaric acid, or a combination thereof. Preferably, the acid is nitric acid.

[00130] Optionally, the residence time of the natural source in the acidic hot water is 2 to 4.5 hours, preferably 2.5 to 4 hours, and preferably 3 to 3.5 hours.

[00131] Preferably, the acidic hot water is at a temperature of 70 to 80 °C, with a pH of 1.90 to 2.10, and the natural source has a residence time in the acidic hot water of 3 to 3.5 hours.

[00132] HM pectin is extracted in an aqueous form. HM pectin in aqueous form can be clarified using centrifugation and filtration.

[00133] Optionally, HM pectin in aqueous form is clarified by centrifugation and then by filtration.

[00134] Once centrifugation and filtration are complete, a filtered aqueous form containing HM pectin is formed. Petition 870250088676, dated 09 / 30 / 2025, page 32 / 52 / 35

[00135] The calcium present in the filtered aqueous form containing HM pectin can be removed using ion exchange. Optionally, a cationic resin is used to perform the calcium removal.

[00136] The calcium removal product is a decalcified aqueous form containing HM pectin. Optionally, the HM pectin in the decalcified aqueous form containing HM pectin comprises 600 ppm of calcium or less, preferably 550 ppm of calcium or less, and most preferably 500 ppm of calcium or less.

[00137] The decalcified aqueous form containing HM pectin can be concentrated by evaporation to form a concentrated HM pectin in aqueous form. Any methods known in the art for concentrating a liquid can be used.

[00138] Optionally, concentrated HM pectin in aqueous form comprises HM pectin of 15 to 40 g / L, preferably 20 to 37 g / L, more preferably 25 to 35 g / L, and most preferably 27 to 30 g / L.

[00139] The degree of esterification (DE) of concentrated HM pectin in aqueous form can be modified by adding an acidic solution to concentrated HM pectin in aqueous form. The resulting product is HM pectin with modified DE in aqueous form.

[00140] Optionally, the degree of esterification (DE) can be modified by adding acid to the concentrated HM pectin in aqueous form. Optionally, the acid is one or more of nitric acid, phosphoric acid, sulfuric acid, citric acid, malic acid, succinic acid, adipic acid, gluconic acid, tartaric acid, fumaric acid, or a combination thereof. Preferably, the acid is nitric acid. Optionally, the acid solution comprises acid in water. Optionally, the acid is 0.05 to 0.15% by weight of the total volume of the acid solution and concentrated HM pectin in aqueous form, or more preferably, 0.06 to 0.12% by weight of the total volume of the acid solution and concentrated HM pectin in aqueous form, or Petition 870250088676, dated 09 / 30 / 2025, p. 33 / 52 / 35 with the highest preference, from 0.7 to 1.1%, by weight, of the total volume of the acidic solution and concentrated HM pectin in aqueous form.

[00141] Optionally, the temperature of the step to modify the degree of esterification (DE) is 20 to 45 °C, preferably 25 to 40 °C, and most preferably 30 to 35 °C.

[00142] Optionally, the residence time of concentrated HM pectin in aqueous form in hot acidic water is 50 to 110 hours, preferably 60 to 105 hours, more preferably 70 to 100 hours, and most preferably 72 to 96 hours.

[00143] Optionally, the resulting product has a degree of esterification (DE) of 40 to 75%, preferably 45 to 70%, more preferably 50 to 65%, and most preferably 54 to 58%.

[00144] Preferably, modifying the degree of esterification (DE) of concentrated HM pectin in aqueous form requires a temperature of 30 to 35 °C, an acid content of 0.70 to 1.10% by weight of the total weight of the mixture, a residence time of the concentrated HM pectin in aqueous form in hot acidic water of 72 to 96 hours, and the resulting product has a degree of esterification (DE) of 54 to 58%.

[00145] An appropriate amount of alcohol is added to the modified DE HM pectin in aqueous form to form an alcohol / water mixture. The amount of alcohol added depends on the amount of alcohol required to cause the HM pectin to precipitate from its aqueous form as a precipitate containing the HM pectin.

[00146] Optionally, the alcohol used is isopropanol, tert-butyl alcohol, benzyl alcohol, 1,4-butanediol, 1,2,4-butanetriol, butanol, 1-butanol, 2-butanol, tert-butyl alcohol, diethylene glycol, ethanol, ethylene glycol, 2-ethylhexanol, furfuryl alcohol, glycerol, isobutanol, isopropyl alcohol, methanol, 2-(2-methoxyethoxy)ethanol, 2-methyl-1-butanol, 2-methyl-1-pentanol, 3-methyl-2-butanol, neopentyl alcohol, 2-pentanol, 1,3-propanediol, propan-1 Petition 870250088676, dated 09 / 30 / 2025, page 34 / 52 / 35 ol, propylene glycol, propylene glycol methyl ether, or any combination thereof. Preferably, the alcohol is isopropyl alcohol.

[00147] The precipitate containing HM pectin may be further washed to remove any impurities present. Optionally, the precipitate containing HM pectin is further washed with alcohol to form a washed HM pectin.

[00148] Optionally, the alcohol used is isopropanol, tert-butyl alcohol, benzyl alcohol, 1,4-butanediol, 1,2,4-butanetriol, butanol, 1-butanol, 2-butanol, tert-butyl alcohol, diethylene glycol, ethanol, ethylene glycol, 2-ethylhexanol, furfuryl alcohol, glycerol, isobutanol, isopropyl alcohol, methanol, 2-(2-methoxyethoxyl)ethanol, 2-methyl-1-butanol, 2-methyl-1-pentanol, 3-methyl-2-butanol, neopentyl alcohol, 2-pentanol, 1,3-propanediol, propan-1-ol, propylene glycol, propylene glycol methyl ether, or any combination thereof. Preferably, the alcohol is isopropyl alcohol.

[00149] Washed HM pectin can be pressed to form wet precipitated HM pectin. Wet precipitated HM pectin has a dry matter content of 20 to 80% by weight, preferably 30 to 70% by weight, more preferably 40 to 60% by weight, even more preferably 45 to 55% by weight, and most preferably 50% by weight of the total weight of wet precipitated HM pectin is achieved. Preferably, wet precipitated HM pectin has a dry matter content of 50% by weight of its total weight.

[00150] Optionally, wet precipitated HM pectin is a slow-setting pectin.

[00151] Optionally, the wet precipitated HM pectin can be further dried to form a dry HM pectin. Optionally, the dry HM pectin is dried to a moisture content of 5 to 15% by weight, preferably 7.5 to 12.5% ​​by weight, and most preferably 10% by weight of the total weight of the dry HM pectin. The dry HM pectin can then, Petition 870250088676, dated 09 / 30 / 2025, page 35 / 52 / 35 to be crushed until the dry HM pectin has a homogeneous particle size distribution and / or is mixed with sugar to form a standardized HM pectin. Step 2: Amidation of precipitated wet HM pectin

[00152] Wet precipitated HM pectin can then be amidated by placing it in a suspension with alcohol and ammonia to form an amidated LM pectin.

[00153] Optionally, the alcohol is any one or more of tert-butyl alcohol, benzyl alcohol, 1,4-butanediol, 1,2,4-butanetriol, butanol, 1-butanol, 2-butanol, tert-butyl alcohol, diethylene glycol, ethanol, ethylene glycol, 2-ethylhexanol, furfuryl alcohol, glycerol, isobutanol, isopropyl alcohol, methanol, 2-(2-methoxyethoxyl)ethanol, 2-methyl-1-butanol, 2-methyl-1-pentanol, 3-methyl-2-butanol, neopentyl alcohol, 2-pentanol, 1,3-propanediol, propan-1-ol, propylene glycol, propylene glycol methyl ether, isopropanol, or any combination thereof. Preferably, the alcohol is isopropyl alcohol.

[00154] Preferably, alcohol is present at 60 to 70% by weight of the total weight of the suspension, ammonia is present at 4 to 6% by weight of the total weight of the suspension, the temperature is 4 to 6 °C and the reaction mixture is maintained for 3 to 6 hours.

[00155] Optionally, alcohol may be present from 50 to 80% by weight, preferably from 55 to 75% by weight, and most preferably from 60 to 70% by weight of the total weight of the suspension. Preferably, alcohol is present from 60 to 70% by weight of the total weight of the suspension.

[00156] Optionally, ammonia is present from 1 to 10% by weight, preferably from 2 to 8% by weight, more preferably from 3 to 7% by weight, and most preferably from 4 to 6% by weight of the total weight of the suspension. Preferably, ammonia is present from 4 to 6% by weight of the total weight of the suspension.

[00157] Optionally, the reaction mixture containing HM pectin Petition 870250088676, dated 09 / 30 / 2025, page 36 / 52 / 35 wet precipitate, alcohol and ammonia is at a temperature of 1 to 12 °C, preferably 2 to 10 °C, more preferably 3 to 8 °C, and most preferably 4 to 6 °C. Preferably, the temperature is 4 to 6 °C.

[00158] Optionally, the reaction mixture is maintained for 1 to 8 hours, preferably for 2 to 7 hours, even more preferably for 2.5 to 6.5 hours, and most preferably for 3 to 6 hours. Preferably, the reaction mixture is maintained for 3 to 6 hours.

[00159] To prevent the reaction from occurring in the reaction mixture, amidated LM pectin can be filtered from the alcohol and ammonia by filtration to produce a filtrate comprising the alcohol and a residue comprising the amidated LM pectin.

[00160] Optionally, the filtrate comprising alcohol and ammonia may undergo a recovery step.

[00161] Optionally, the residue comprising amidated LM pectin may be neutralized to prevent further reaction to form a neutralized amidated LM pectin.

[00162] Optionally, the reaction is neutralized with an acidulated alcohol. Optionally, the acidulated alcohol comprises a mineral acid. Optionally, the mineral acid is nitric acid. Optionally, the acidulated alcohol further comprises any of the following: tert-butyl alcohol, benzyl alcohol, 1,4-butanediol, 1,2,4-butanetriol, butanol, 1-butanol, 2-butanol, tert-butyl alcohol, diethylene glycol, ethanol, ethylene glycol, 2-ethylhexanol, furfuryl alcohol, glycerol, isobutanol, isopropyl alcohol, methanol, 2-(2-methoxyethoxy)ethanol, 2-methyl-1-butanol, 2-methyl-1-pentanol, 3-methyl-2-butanol, neopentyl alcohol, 2-pentanol, 1,3-propanediol, propan-1-ol, propylene glycol, propylene glycol methyl ether, isopropanol, or any combination thereof. Preferably, the alcohol is isopropyl alcohol.

[00163] Optionally, the residue comprising amidated LM pectin is neutralized with acidulated alcohol until the pH of the LM pectin Petition 870250088676, dated 09 / 30 / 2025, page 37 / 52 / 35 amidated neutralized is from 3.0 to 5.0, preferably from 4.0 to 5.0, and most preferably from 4.0 to 4.5. Preferably, the residue comprising the amidated LM pectin is neutralized with an acidulated alcohol comprising nitric acid and isopropyl alcohol until the neutralized amidated LM pectin has a pH of 4.0 to 4.5.

[00164] Neutralized amidated LM pectin can be washed off with alcohol.

[00165] Optionally, the alcohol is any of the following: tert-butyl alcohol, benzyl alcohol, 1,4-butanediol, 1,2,4-butanetriol, butanol, 1-butanol, 2-butanol, tert-butyl alcohol, diethylene glycol, ethanol, ethylene glycol, 2-ethylhexanol, furfuryl alcohol, glycerol, isobutanol, isopropyl alcohol, methanol, 2-(2-methoxyethoxy)ethanol, 2-methyl-1-butanol, 2-methyl-1-pentanol, 3-methyl-2-butanol, neopentyl alcohol, 2-pentanol, 1,3-propanediol, propan-1-ol, propylene glycol, propylene glycol methyl ether, isopropanol, or any combination thereof. Preferably, the alcohol is isopropyl alcohol.

[00166] Optionally, the neutralized amidated LM pectin is washed with alcohol one, two, three, four, five, six, seven, eight, nine, ten or more than ten times. Preferably, the neutralized amidated LM pectin is washed with alcohol three times.

[00167] After the neutralized amidated LM pectin is washed, the neutralized amidated LM pectin can be subjected to a pressing step. The product of the pressing step is a pressed amidated LM pectin.

[00168] Optionally, the amidated neutralized LM pectin can be pressed until the product of the pressing step has a dry substance of 30 to 70% by weight, preferably 40 to 60% by weight, more preferably 45 to 55% by weight, and most preferably 50% by weight, of the total weight of the product of the pressing step.

[00169] Pressed amidated LM pectin can be subjected to a drying step. The product of the drying step is LM pectin. Petition 870250088676, dated 09 / 30 / 2025, page 38 / 52 / 35 amended.

[00170] Optionally, the drying step removes water and / or alcohol present in the pressed amidated LM pectin.

[00171] Optionally, the pressed amidated LM pectin can be dried until the product of the drying step has a moisture content of 1 to 30% by weight, preferably 2 to 20% by weight, more preferably 7.5 to 15.0% by weight, even more preferably 9 to 11% by weight, and most preferably 10% by weight of the product of the drying step.

[00172] The amidated LM pectin is then ready for quality analysis and further modification. Further modifications may include standardization with sugar to make a commercial product and / or grinding the amidated LM pectin. Amidated LM pectin in a food or beverage product.

[00173] Another aspect of the present invention relates to an amidated LM pectin in a food product or beverage.

[00174] Amidated LM pectin can be incorporated into a dairy product, which includes, but is not limited to, milk, condensed milk, dried milk, dairy drinks, cream, butter, yogurt, cheese, custard, cottage cheese, cream cheese, curd, frozen dairy products such as frozen custard, frozen yogurt and ice cream, gelato, milk powder, evaporated milk, curdled milk, whey, whey protein or whipped cream. Method for producing amidated LM pectin in a food or beverage product.

[00175] Another aspect of the present invention relates to a method for producing a food product or beverage comprising amidated LM pectin. The method for producing the food product or beverage comprises the steps of: (a) provide amidated LM pectin; and (b) combine amidated LM pectin with at least one Petition 870250088676, dated 09 / 30 / 2025, p. 39 / 52 / 35 food product or beverage. Examples

[00176] The following are non-limiting examples which discuss, with reference to tables and figures, the advantages of the present invention. The examples presented in this document are merely examples among other possible examples. Example 1: A non-limiting example of the formation of an amidated LMA pectin by the wet amidation process.

[00177] In this non-limiting example, an amidated LM pectin was produced by the wet amidation method according to the first aspect of this disclosure. In this example, the amidated LM pectin was obtained by (a) extracting HM pectin from a citrus fruit peel to form a wet precipitated HM pectin and (b) forming an amidated LM pectin by subjecting the wet precipitated HM pectin to amidation.

[00178] In this non-limiting example, the following steps were taken to (a) extract HM pectin from a citrus fruit peel to form a wet precipitated HM pectin: 1. Mix citrus peel with hot acidic water, ensuring the temperature of the hot acidic water is 70 to 80 °C, the pH is 1.90 to 2.10, and the residence time is 3 to 3.5 hours.

[00179] 2. Clarify the product from step 1 using centrifugation and filtration.

[00180] 3. Use a cationic resin to remove calcium from the product from step 2.

[00181] 4. Concentrate the product from step 3 until HM pectin is present in water at a concentration of 27 to 30 g / L.

[00182] 5. Modify the degree of esterification (DE) of the product of the step by adding nitric acid. The step is conducted at a temperature of 30 to 35 °C, the nitric acid is present at 0.80 to 1.0%, by weight, of the total weight of the Petition 870250088676, dated 09 / 30 / 2025, page 40 / 52 / 35 mixture, and the residence time is 72 to 96 hours. The resulting product has a degree of esterification (DE) of 54 to 58%. The resulting degree of esterification is measured by titration.

[00183] 6. Precipitate the HM pectin from the product of step 5 by adding isopropyl alcohol.

[00184] 7. Wash the product from step 6 with isopropyl alcohol.

[00185] 8. Press the product from step 7 until the product from step 7 has a dry substance that is 50% by weight of the total weight of the product from the drying step. The product is a wet precipitated HM pectin.

[00186] In this non-limiting example, the following steps were taken to form an amidated LM pectin by subjecting wet precipitated HM pectin to amidation.

[00187] 9. Mix the precipitated wet HM pectin with isopropyl alcohol and ammonia. The alcohol is present at 60 to 70% by weight of the total weight of the mixture, the ammonia is present at 4.0 to 6.0% by weight of the total weight of the mixture, and the suspension is maintained at a temperature of 4 to 6 °C. The product of step 8 is in suspension for 3 to 6 hours. The reaction converts the HM pectin into an amidated LM pectin.

[00188] 10. Filter the amidated LM pectin formed in step 9 from the isopropyl alcohol and ammonia.

[00189] 11. Neutralize the amidated LM pectin with an acidulated alcohol, wherein the acidulated alcohol comprises nitric acid in isopropyl alcohol.

[00190] 12. Wash the product from step 11 with isopropyl alcohol three times.

[00191] 13. Press the product from step 12 until the product from that step has a dry substance that is 50%, by weight, of the total weight of the product from the pressing step.

[00192] 14. Dry the product from step 13 until the product from step 13 Petition 870250088676, dated 09 / 30 / 2025, page 41 / 52 / 35 has a moisture content of 10%, by weight, of the total weight of the product from the drying stage. The resulting product is amidated LM pectin.

[00193] In step 5, the degree of esterification resulting from HM pectin is measured by titration. HM pectin is washed with acidified isopropanol (IPA) to remove any cations, salts, and sugar present before titration. Then, the titration is performed using 0.1% bromothymol blue in ethanol as the indicator. The purified HM pectin is mixed with five drops of bromothymol blue indicator and then titrated with 0.1 mol / L sodium hydroxide. The ester groups of HM pectin are saponified with 0.5 mol / L sodium hydroxide, which is then neutralized with exactly the same amount of 0.5 mol / L hydrochloric acid, so that the released acidic groups can then be titrated with 0.1 mol / L sodium hydroxide solution. The method described is an example of a JECFA procedure. Example 2: A non-limiting example of comparing the intrinsic viscosity of an amidated LM pectin formed through the wet amidation process according to the traditional methods 1, 2 and 3 set out above with the amidated LM pectin formed through the wet amidation method of the present invention.

[00194] In this non-limiting example, the intrinsic viscosity of LM pectin before amidation was compared with amidated LM pectins as produced by the traditional methods 1, 2 and 3 set out above, and amidated LM pectin produced by the method set out in example 1.

[00195] In this non-limiting example, a dry HM pectin and an amidated LM pectin produced using the traditional method 1 were compared. The dry HM pectin is referred to as dry HM pectin (1) and the amidated LM pectin is referred to as amidated LM pectin (1) hereafter. The dry HM pectin (1) and the amidated LM pectin (1) were produced using the method set out under the heading “Traditional Method 1”, wherein Petition 870250088676, dated 09 / 30 / 2025, page 42 / 52 / 35 dry HM pectin (1) was the product of step (1) of the method and amidated LM pectin (1) was the product of step (2) of the method.

[00196] In this non-limiting example, a dry HM pectin and an amidated LM pectin, produced using the traditional method 2, were also compared. The dry HM pectin is referred to as dry HM pectin (2) and the amidated LM pectin is referred to as amidated LM pectin (2) hereafter. The dry HM pectin (2) and the amidated LM pectin (2) were produced using the method set out under the heading “Traditional Method 2”, wherein the dry HM pectin (2) was the product of step (1) of the method and the amidated LM pectin (2) was the product of step (2) of the method.

[00197] In this non-limiting example, a dry HM pectin and an amidated LM pectin, produced using the traditional method 3, were also compared. The dry HM pectin is referred to as dry HM pectin (3) and the amidated LM pectin is referred to as amidated LM pectin (3) hereafter. The dry HM pectin (3) is the same as the dry HM pectin (2). The dry HM pectin (3) and the amidated LM pectin (3) were produced using the method established under the heading “Traditional Method 3”, wherein the dry HM pectin (3) was the product of step (1) of the method and the amidated LM pectin (3) was the product of step (2) of the method.

[00198] In this non-limiting example, a wet precipitated HM pectin and an amidated LM pectin, produced using the method according to the present disclosure, were also compared. The wet precipitated HM pectin is referred to as wet precipitated HM pectin (4) and the amidated LM pectin is referred to as amidated LM pectin (4) hereafter. The wet precipitated HM pectin (4) and the amidated LM pectin (4) were produced using the method set out in Example 1, wherein the wet precipitated HM pectin (4) was the product of step (8) of the method and the amidated LM pectin (4) was the product of step (14) of the method.

[00199] The intrinsic viscosity of each of the dry HM pectins Petition 870250088676, dated 09 / 30 / 2025, page 43 / 52 / 35 (1), amidated LM pectin (1), dry HM pectin (2), amidated LM pectin (2), dry HM pectin (3), amidated HM pectin (3), wet precipitated HM pectin (4) and amidated HM pectin (4) was measured. Intrinsic viscosity measurements were performed using a gel permeation chromatography (GPC) system equipped with a Viscotek TDA instrument with multiple detectors (detectors being refractive index, right-angle laser scattering (90°) and low-angle laser scattering (9°) and a viscometer). To determine the intrinsic viscosity, each sample was dissolved separately at a concentration of 2 mg / mL and eluted with a 0.1 M LiNO3 buffer solution and 1 g / L EDTA. After complete dissolution, each sample was filtered separately through 0.2 micrometer porosity membranes: the injection volume was 100 microliters and the flow rate was 0.75 mL / min.The chosen analytical method was the triple point method, calibrated with pullulan supplied by the equipment vendor, with the MP (molecular weight of the highest peak) ranging between 5,000 and 250,000 g / mol. Measurements were taken at 35 °C.

[00200] The results of the intrinsic viscosity analysis are shown in Table 1 and Figure 4. Table 1: The intrinsic viscosity values ​​of HM pectin and amidated LM pectin, produced using traditional methods 1 to 3 and the method established in Example 1. Sample Intrinsic Viscosity (mL / g) Difference in intrinsic viscosity between dry HM pectin and amidated LM pectin (mL / g)* Dry HM pectin (1) 340 to 427 Approximately 67 Amidated LM pectin (1) 267 to 341 Dry HM pectin (2) 453 to 594 Approximately 142 Amidated LM pectin (2) 325 to 393 Dry HM pectin (3) 453 to 594 Approximately 102 Amidated LM pectin (3) 375 to 425 Wet precipitated HM pectin (4) 525 to 615 Approximately 23 Petition 870250088676, dated 09 / 30 / 2025, page 44 / 52 / 35 Amidated LM pectin (4) 488 to 600 * These values ​​were determined from the average intrinsic viscosity difference for each sample.

[00201] As can be seen in Table 1 and Figure 4, the difference in intrinsic viscosity values ​​for HM pectin (4) and amidated LM pectin (4) is smaller than for HM pectins and amidated LM pectins made according to traditional methods. Advantageously, pectin is subjected to much less degradation when subjected to the amidated pectin formation method according to the present invention and, therefore, the intrinsic viscosity values ​​are much closer. Petition 870250088676, dated 09 / 30 / 2025, pp. 45 / 52

Claims

1 / 2 CLAIMS 1. Method for producing a low-methoxyl amidated pectin, the method characterized in that it comprises the following step: adding a wet precipitated high-methoxyl pectin to a suspension comprising one or more alcohols and ammonia to form a low-methoxyl amidated pectin.

2. Method according to claim 1, characterized in that the low-methoxylated amidated pectin has a degree of methylation below 50%.

3. Method according to claim 1 or claim 2, characterized in that the precipitated wet pectin of high methoxylation has a degree of methylation above 50%.

4. Method according to any one of claims 1 to 3, characterized in that the wet precipitated pectin of high methoxylation has an intrinsic viscosity of 500 to 650 mL / g, or 510 to 640 mL / g, or 525 to 615 mL / g.

5. Method according to any one of claims 1 to 4, characterized in that the low methoxylated amidated pectin has an intrinsic viscosity of 450 to 650 mL / g, or 475 to 625 mL / g, or 488 to 600 mL / g.

6. Method according to any one of claims 1 to 5, characterized in that wet precipitated pectin of high methoxylation is in suspension with one or more alcohols and ammonia at a temperature of 1 to 12 °C, or 2 to 10 °C, or 3 to 8 °C, or 4 to 6 °C.

7. Method according to any one of claims 1 to 6, characterized in that wet precipitated pectin of high methoxylation is in suspension with one or more alcohols and ammonia for a reaction time of 1 to 8 hours, or 2 to 7 hours, or 2.5 to 6.5 hours, or Petition 870250088676, dated 09 / 30 / 2025, page 46 / 52 2 / 2 3 to 6 hours.

8. A method according to any one of claims 1 to 7, characterized in that the wet precipitated pectin of high methoxylation has a dry matter content of 20 to 80% by weight, or 30 to 70% by weight, or 40 to 60% by weight, or 45 to 55% by weight, or 50% by weight, of the total weight of the wet precipitated pectin of high methoxylation.

9. A method according to any one of claims 1 to 8, characterized in that ammonia is present in the suspension at 1 to 10% by weight, or 2 to 8% by weight, or 3 to 7% by weight, or 4 to 6% by weight, of the total weight of the suspension.

10. A method according to any one of claims 1 to 9, characterized in that one or more alcohols are present in the suspension at 50 to 80% by weight, or 55 to 75% by weight, or 60 to 70% by weight of the total weight of the suspension.

11. A method according to any one of claims 1 to 10, characterized in that one or more alcohols are any one of tert-amyl alcohol, benzyl alcohol, 1,4-butanediol, 1,2,4-butanetriol, butanol, 1-butanol, 2-butanol, tert-butyl alcohol, diethylene glycol, ethanol, ethylene glycol, 2-ethylhexanol, furfuryl alcohol, glycerol, isobutanol, isopropyl alcohol, methanol, 2-(2-methoxyethoxy)ethanol, 2-methyl-1-butanol, 2-methyl-1-pentanol, 3-methyl-2-butanol, neopentyl alcohol, 2-pentanol, 1,3-propanediol, propan-1-ol, propylene glycol, propylene glycol methyl ether, isopropanol, or any combination thereof. Petition 870250088676, dated 09 / 30 / 2025, pages 47 / 52

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