Pectin characterized by low degree of methyl esterification and high intrinsic viscosity

Abstract

To provide citrus pectin with a low degree of methyl esterification and a high intrinsic viscosity, a process for producing the same, and use of an enzyme for producing pectin.SOLUTION: A citrus pectin is provided having: a) a degree of methyl esterification of 30 or less; and b) an intrinsic viscosity of 5 dl / g or more. A de-esterification process for producing the citrus pectin comprises incubating a citrus peel in a solution comprising: (a) a buffer which maintains the pH of the solution containing the citrus peel at a pH of 5 to 8; and (b) a calcium binder.SELECTED DRAWING: Figure 1B

Description

[Technical field]

[0001] The present invention relates to a citrus pectin which has a low degree of methyl esterification but a high intrinsic viscosity. A method for producing pectin using citrus peels, and a method for producing pectin that is inherent in citrus peels and that and the use of the enzyme to produce cyclohexyl ether. [Background technology]

[0002] Pectin is an important polysaccharide with applications in the food, pharmaceutical, and various other industries. The polysaccharide chain contains large amounts of substituted α1→4-linked anhydrogalacturonic acid (AGU). The substitution refers to a methyl esterification at the 6-position (Figure 1b). The proportion of carboxyl groups that have been methyl-esterified at this position is called the "degree of methyl esterification" or The abbreviation is DM. Commercially available pectin has a high methyl ester content if its DM is 50% or higher. This is commonly called HM pectin. Pectins with a DM of less than 50 Pectin is commonly referred to as low methyl ester pectin or LM pectin.

[0003] The DM value strongly influences how pectin interacts with other substances in solution. Cutin has a large influence on the food applications it can be used in. For example, the sucrose content Jams with a sucrose content of about 65% are typically gelled with HM pectin, but with a sucrose content of 5 LM pectin is preferred for jams with less than 5%. Another example: HM pectin is preferred for yogurt drinks. Although LM pectin is often used in food preparations, it is usually used in traditional yogurt (eating with a spoon). (which can be done) is preferable.

[0004] In addition to DM, intrinsic viscosity (IV) is also an important value in determining the use of pectin. V is the ability of a substance to thicken a solvent. Pectin is a pectin polymer. A high IV can be achieved by maintaining a high number of repeating sugar units within the oligosaccharide.

[0005] The DM of pectin determines its solubility and the ratio of the solvent to the pectin. The IV of pectin depends on the amount of pectin and the quality of the food. and the robustness of the desired effect. Higher IV pectins are less prone to instability. It is capable of producing strong, elastic gels. Similarly, high IV pectins are homogeneous. Has a hazy or "milky" appearance and does not easily exude a thin liquid or produce a visible precipitate The present invention can produce stable milk protein suspensions that do not form viscous or viscous fluids.

[0006] Traditional pectin extraction involves acidic conditions, for example using strong mineral acids such as nitric acid. However, to maintain a high IV, mild conditions such as pH 2.4 and a temperature of 70°C were used. Over time, the yield decreases. To ensure a relatively high IV while still obtaining satisfactory yields. When extracted by the procedure, the DM of the pectin is at least about 55, and typically higher. To reduce DM, pectin can be incubated with strong acid or alkali. This allows the chlorine to be removed by hydrolysis (in the case of acids) or saponification (in the case of alkalis). The methyl group is separated from the carboxyl group. However, in addition to cleaving the ester, Alternatively, strong alkali may degrade the main polysaccharide chain, causing a decrease in IV. This will result in a lower DM at the expense of...

[0007] Alternatively, pectin can be incubated with a microbial pectinesterase enzyme. This allows for a reduction in DM compared to acid or alkaline deesterification. However, the microbial pectinesterase enzyme may be produced, resulting in a smaller loss of IV. To be of any use, pectin must first be extracted from the peel. The enzymes (which should not act in the final pectin) are activated by heating the mixture to high temperatures. Therefore, it must be inactivated, which also reduces the IV. Summary of the Invention

[0008] The inventors have developed a method to produce pectin with low DM while maintaining a high IV. .

[0009] Thus, the present invention provides a) the degree of methyl esterification is 30 or less; b) Intrinsic viscosity is 5 dl / g or more The present invention provides a citrus pectin characterized by:

[0010] Pectin has a degree of methyl esterification of It can be a) 20 or less; b) 15 or less; or c) 10 or less.

[0011] Citrus pectin is pectin derived from oranges, lemons, grapefruits or limes. It could be.

[0012] The intrinsic viscosity of the pectin can be: a) 6 dl / g or more; or b) 7 dl / g or more.

[0013] The present invention also relates to a methyl esterification degree of 30 or less; and an intrinsic viscosity of 5 dl / g or less. The present invention provides a method for producing pectin, characterized in that The sterilization method comprises the steps of incubating citrus peels in a solution comprising: a) a buffer that maintains the pH of the solution containing the peel at pH 5-8; b) Calcium binders.

[0014] The resulting pectin produced by this method has, as described above, a) methyl ether a) a degree of esterification of 30 or less; and b) a citrus pectin having an intrinsic viscosity of 5 dl / g or more. It is possible.

[0015] The incubation period may be 30 minutes or more.

[0016] It is endogenous to citrus peels and is used as an enzyme to de-esterify pectins in citrus peels. The deesterification reaction is carried out in a) a buffer that maintains the pH between 5 and 8, and b) a calcium salt. and a sodium binder.

[0017] The following description applies to either the deesterification reaction of the method or the use of the method.

[0018] The concentration of the buffer may be 0.02M or greater.

[0019] The temperature of the deesterification reaction may be: a) 55°C to 75°C; or b) 59°C to 65°C. do.

[0020] The calcium-binding agent may also act as a buffer. For example, a calcium-binding buffer may be a) Na2H-citrate; b) Na3-citrate; c) Na2H-phosphate; or d) N It can be a3-phosphate.

[0021] Alternatively, or additionally, the calcium binding agent may comprise cation exchange beads.

[0022] The citrus peel may be orange, lemon or lime peel.

[0023] There is also provided a pectin obtainable by the above process.

[0024] Throughout this specification, unless the context requires otherwise, the term "comprising" is used. e)" or "include" or "comprises " or "comprising" or "includes" Variations such as "including" or "including" refer to the method or kit. is meant to contain the stated integer or group of integers, but may contain any other integer or group of integers. It will be understood that this is not meant to be exclusionary.

[0025] Each document, reference, patent application or patent cited within this text is hereby incorporated by reference in its entirety. It is expressly incorporated herein and should be read and considered by the reader as part of the text. This means that documents, references, patent applications, or patents cited in the text are included in the text. It is for the sake of brevity only that the references and Any reference to information indicates that the material or information was part of the general knowledge or was available in any country. This document should not be construed as an admission that any of the following was previously known:

[0026] The invention will now be further described, by way of example only, with reference to the accompanying examples. [Brief description of the drawings]

[0027] [Figure 1A] FIG. 1 shows regions of substituted α1→4 linked anhydrogalacturonic acid in pectin. [Figure 1B] FIG. 1 shows position 6 where the substitution occurs. [Diagram 2]FIG. 1 shows the IV and DM of pectins (circles) made by the described novel process. Pectins prepared by conventional acid deesterification are represented by diamonds, and pectins prepared by prior art enzymatic deesterification are represented by triangles. The open circles are 18 liter scale production, the small circles with slashes represent 36 liter scale, and the large filled circles represent 800 liter scale examples from Tables 2 (black fill) and 3 (grey fill). The rectangles (solid lines) delimit the parameter space of enzymatically deesterified pectins as described in Hoejgaard et al, Pectin for Heat Stable Bakery Jams (US20070621747 20070110). The claimed composition is in the upper left corner within the dotted line. [Diagram 3] Figure 1 shows the IV and yield of pectins made by the described novel process. Prior art pectin LMC: chemically de-esterified low DM pectin (H: open diamonds below the dashed line at PVIV 100) is included for comparison. The abbreviations A-G represent different peel producers. [Figure 4] FIG. 13 shows the MODDE coefficients in the linear models of apparent η=f(DM, IV, pH) and apparent G′=f(DM, IV, pH). [Diagram 5] FIG. 1 shows "silver pearls" (e.g. for cake decoration) suspended in an acidified pectin solution. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] (Pectin) Pectin contains large regions of substituted α1→4-linked anhydrogalacturonic acid (AGU). Pectins from different fruits, vegetables and plants have different types For example, aloe leaf pectin contains sugars not found in citrus pectins. It contains 3-OMe-rhamnose, a rare sugar that is not found in different fruits, vegetables and plants. Pectins from different sources also differ in the proportions of different sugars they contain. Ngopectin has a much higher xylose content than citrus pectins.

[0029] As used herein, the term pectin includes pectin salts, also known as pectates. The pectins described herein are understood by those of skill in the art given the conventional definition of pectin. As such, it is a nonamidated pectin.

[0030] (Methyl esterification degree) The ratio of methyl-esterified carboxyl groups in pectin is called the "degree of methyl esterification" or It is abbreviated as DM.

[0031] The degree of methyl esterification of the pectin may be 30 or less. For example, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 1 The degree of methyl esterification is not more than 3, 12, 11, 10, 9, 8, 7, 6, or 5. The range may include any of the following:

[0032] The degree of methyl esterification can be measured according to the protocol described in the Examples section. .

[0033] For example, by titration with an indicator.

[0034] (intrinsic viscosity) The IV of a dissolved polymer (e.g., pectin) is determined not only by its composition and covalent structure, but also by the The more one of these molecules "feels completely alone," the more likely it is to react with the solvent. IV is explained as the inherent ability of a substance to thicken a solvent when dissolved in low concentrations. A more precise definition is provided by the IUPAC organization and can be found here To summarize: if η is the viscosity of a solution of a substance and the concentration of said substance is called c, then A series of solutions of various concentrations (c) were presented and their corresponding viscosities (η) were measured. If we assume that the viscosity of the pure solvent is η0, the viscosity of the substance in the solvent is The viscosity is IV=lim c→0 It is given by ((η-η0) / (c η0)). The "lim c→0 " refers to the various values ​​from c to c = 0 ( This means that the value of (η-η0) / (c η0) is extrapolated. The dimension of IV is the inverse of the concentration and values ​​are often reported in units of dl / g.

[0035] The classical method for determining the intrinsic viscosity (IV) of pectin is to measure several solutions of pectin. One method is to prepare solutions of these materials and measure their viscosities using a viscometer.

[0036] More modern techniques of liquid chromatography can also be used. It is possible to use a chromatograph equipped with a viscosity detector as well as a detector that quantifies concentration. Chromatography of pectin reveals a series of relationships between pectin concentration and viscosity. The IV can then be interpreted as a statistical population, with the average The IV can be calculated.

[0037] The IV referred to in this application is a method for determining whether the IV is a chromatographic method, such as liquid chromatography, e.g., gel permeation chromatography. The average IV is calculated by the averager method. The mean of the values ​​obtained is shown in the table.

[0038] The eluent for which the viscosity is measured may be one used for chromatography. An example of an eluent is lithium acetate. The eluent is used at a concentration of about 0.3M. For example, 0.1, 0.2, 0.4, 0.5 or 0.6M.

[0039] The pH of the eluent may be around 4.6. For example, 3.5 to 5.5. For example, 3. 5, 4, 4.5, 5 or 5.5, or any range of these values.

[0040] The concentration of pectin may range from 0.5 mg to 1.5 mg / ml. For example, The concentration is / ml.

[0041] The temperature may be kept constant at 37°C ± 1°C.

[0042] For example, viscosity was measured at 37°C using 0.3 M lithium acetate at pH 4.6 as the solvent. It can be measured.

[0043] A detailed protocol for determining the IV is provided in the Examples section below.

[0044] The IV can be 5 dl / g or more. For example, the IV can be 5.5 dl / g, 5.6, 5.7 , 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6 .8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8 , 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8 .9, or greater than 9 dl / g.

[0045] (method) The method for producing pectin with a DM of 30 or less and an IV of 5 dl / g or more is to treat citrus peels with a) incubating the solution containing the peel in a solution containing pH 5 a) a buffer that maintains the temperature at ∼8; and b) a calcium-binding agent.

[0046] The inventors have demonstrated that pH is a key factor in determining whether endogenous citrus peel pectin can be de-esterified. The hypothesis is that it strongly influences enzymes (endogenous enzymes are those naturally present in citrus peels). Deesterification refers to the enzyme that is present in the The present inventors further demonstrated that the calcium-binding agent can bind calcium from pectin. By removing the methyl group, the endogenous enzyme can access the ester bond and cleave the methyl group. It is hypothesized that pectin is de-esterified to produce LM-pectin. The resulting de-esterified pectin is released into solution. It is not necessary to first extract the pectin (e.g., with acid) prior to esterification. is deesterified in situ in fruit or vegetable biomass and released into solution The in situ deesterification conditions are mild and do not inhibit the activity of endogenous enzymes. Therefore, the resulting LM-pectin also exhibits a high IV value.

[0047] Therefore, the deesterification conditions to stabilize the enzyme are highly alkaline or acidic. Deesterification of pectin by alkaline saponification or acid hydrolysis requiring various pH and high temperatures. Thus, the present method is different from that previously used for deesterification of pectin. The extreme temperatures and pH used in polymerization lower the IV of pectin. No alkaline saponification or acid hydrolysis step is required for deesterification (saponification is generally Deesterification with acid generally requires a pH of at least 9. For example, a pH of less than about 1.5, 2, 2.5 or 3.

[0048] The neutralization step is the acid hydrolysis of deesterification to bring the pH back from extreme acidity or alkalinity. It is also often used in the decomposition and alkaline saponification processes. In the present invention, this neutralization step is not necessary since the method is carried out at neutral pH.

[0049] This novel method using endogenous enzymes also overcomes the drawbacks encountered when using microbial enzymes. 1) Catalyzes the de-esterification of pectin, removing it from insoluble plant material into solution. The pectin is first extracted from the skin, since the reaction with the peel enzymes released into the fruit takes place in the skin. 2) the enzymes remain in the peel, which is then removed from the solution, e.g. by a drum vacuum filter; Because the enzyme is filtered out of the solution, there is no need to inactivate it after it has performed its catalytic function. .

[0050] Other pectin-containing biomass may be added to the solution, and the pectin-containing biomass extracted from this biomass may be Pectin is also the result of enzymes from citrus peels. The resulting pectin may be a citrus pectin as described above. The pectin produced by the method described in the specification has a degree of methyl esterification of 30 or less. and b) an intrinsic viscosity of 5 dl / g or more. could be.

[0051] (Buffers and pH) A buffer is a solution that is resistant to changes in pH.

[0052] The preferred pH for the method of producing pectin described herein is pH 5 to 8. For example, pH, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5 .9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9 , 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or The pH can be in any range of the individual pH values ​​listed. For example, pH 5 or less than pH 5-8. For example, 5-7.6, or 5-7.7, or 5 ~7.8, or 5 to 7.9.

[0053] Buffers that work in this pH range include sodium acetate, NaH-citrate or Na 3-Citrate, Na2H-phosphate, NaH2PO4 or Na3-phosphate. Any combination of buffers may be used.

[0054] The buffer(s) used in the method are adapted to maintain the pH within a pH range of 5 to 8. or any other range derived from the individual values ​​above. The pH of the solution to which the peels have been added is adjusted so that the degree of methyl esterification is 30 or less and the IV is 5 dl / g or more. For example, any of the following incubations may be used: Maintain the pH of the solution within a pH range of 5-8 during the incubation period.

[0055] The buffer(s) may be used at a concentration of 0.01M to 0.5M. For example, 0.01M, 0.015M, 0.02M, 0.025M, 0.03M, 0.035M, 0 .04M, 0.045M, 0.05M, 0.055M, 0.06M, 0.065M, 0. 07M, 0.075M, 0.08M, 0.085M, 0.09M, 0.095M, 0.1 M, 0.105M, 0.110M, 0.115M, 0.12M, 0.125M, 0.13 M, 0.135M, 0.14M, 0.145M, 0.15M, 0.155M, 0.16M , 0.165M, 0.17M, 0.175M, 0.18M, ​​0.185M, 0.19M, 0.195M, 0.2M, 0.205M, 0.21M, 0.215M, 0.22M, 0. 225M, 0.23M, 0.235M, 0.24M, 0.245M, 0.25M, 0.3 M, 0.35M, 0.4M, 0.45M, or 0.5M; or from any of these values Any range that can be derived.

[0056] As explained above, the present inventors have demonstrated that buffers stabilize pH-dependent enzymes in citrus peels. The hypothesis is that

[0057] (Calcium binder) Adding the citrus peel enzyme(s) that catalyzes the deesterification to a buffer that stabilizes the enzyme(s) In addition, the method requires a calcium-binding agent.

[0058] As explained above, the present inventors have found that calcium-binding agents bind calcium from pectin in the peel. The sodium is removed, allowing the deesterification enzyme to access the ester bond and catalyze the deesterification reaction. The hypothesis is that this will enable the transfer of

[0059] The calcium-binding agent may be sulfuric acid or a sulfate-containing salt; or oxalic acid or an oxalate salt. It may be a containing salt.

[0060] Preferably, the calcium binding agent also buffers the method to maintain a pH of 5-8. In this way, the calcium binding buffer buffers the process to inhibit the de-esterification of the peel. and to allow the deesterification enzyme to access the pectin in the peel. It is necessary to add calcium to the peel to achieve both functions: There is only one solution. Therefore, the calcium binder is a phosphate or citrate buffer. The cation can be monovalent. Monovalent cations aid in the dissolution of pectin. For example, Na+ or K+. The present inventors have also found that cations such as Na+ and K+ are also soluble in citrus fruits. They hypothesize that this may help activate enzymes in the skin of sea cucumbers.

[0061] For example, Na2H-citrate or Na3-citrate, or combinations of these salts. For example, Na2H-phosphate, NaH2PO4 or Na3-phosphate.

[0062] Alternatively, the calcium binding buffer is an EDTA buffer, such as Na-EDTA; or It may be Na-pyrophosphate.

[0063] The calcium binding buffer can be a combination of these buffers or contain other calcium It may be a binding buffer.

[0064] The calcium-binding agent may be in solution that is added to the peel. Alternatively, the calcium-binding agent may be in solution that is added to the peel. It may also be attached to beads that are added to the peel. For example, cation exchange beads. The sugar is added to a buffer, such as a buffer described herein or above under "Buffers and pH." It can be used in combination with any of the buffer solutions. The binding buffer may be used at a concentration of 0.01M to 0.5M. .015M, 0.02M, 0.025M, 0.03M, 0.035M, 0.04M, 0. 045M, 0.05M, 0.055M, 0.06M, 0.065M, 0.07M, 0.0 75M, 0.08M, 0.085M, 0.09M, 0.095M, 0.1M, 0.105 M, 0.110M, 0.115M, 0.12M, 0.125M, 0.13M, 0.135 M, 0.14M, 0.145M, 0.15M, 0.155M, 0.16M, 0.165M , 0.17M, 0.175M, 0.18M, ​​0.185M, 0.19M, 0.195M, 0.2M, 0.205M, 0.21M, 0.215M, 0.22M, 0.225M, 0. 23M, 0.235M, 0.24M, 0.245M, 0.25M, 0.3M, 0.35M , 0.4M, 0.45M or 0.5M; or any derivation of these values. range. Higher concentrations can be used. However, the lower concentrations mentioned above reduce costs. If the buffer is reused, a higher concentration can be used to balance costs. .

[0065] The buffer concentration is the concentration before adding the peel. For example, 529.4 g of Na3-Cl Add enoic acid, 2·H2O, to 36 L of deionized water. The peel is also added, but only a small amount of the peel is added. The small amount of water is ignored. The peel is not expected to affect the volume of the liquid. The formula mass of enoic acid, 2 H2O, is 294.10, so the result is: Concentration = (529.4 / 29 4.10) / 36M=0.050M=50mM.

[0066] (temperature) The method can be carried out at a temperature of 30 to 75° C. For example, 30, 32, 34, 35, 36, 38, 40, 42, 44, 45, 46, 48, 50, 52, 54, 55, 5 6, 58, 60, 62, 64, 65, 66, 68, 70, 72, 74 or 75℃ or any range of temperatures derived from these values. For example, 55-75°C or 59-65°C. °C.

[0067] (Incubation period) The period of incubation of the peel in the solution may be 20 minutes or more. For example, 30 Minutes or more, 45 minutes or more, 60 minutes or more, 75 minutes or more, 90 minutes or more, 105 minutes or more, 120 minutes or more Upper, 135 minutes or more, 150 minutes or more, 165 minutes or more, 180 minutes or more, 195 minutes or more, 210 225 minutes or more, 240 minutes or more, 255 minutes or more, 270 minutes or more, 285 minutes or more is over 300 minutes.

[0068] (Ingredients of the method) The raw materials used for the extraction of pectin include citrus peels. Citrus peels include orange, lemon, and other fruits. May be mont mongare and / or lime zest.

[0069] (Uses of pectin) The resulting pectin can be used in foods and beverages. When suspending in a gel. EXAMPLES

[0070] Aspects and embodiments of the present invention will now be described, by way of example only, with reference to the following experiments. explain.

[0071] (Protocol for measuring IV) (Orange, lime and lemon by FIPA (Flow Injection Polymer Analysis) Measurement of intrinsic viscosity (IV) of polysaccharide-based pectin

[0072] (principle) The molecules present in the juice were analyzed by gel permeation size exclusion chromatography to determine their size. In the FIPA setup, the pore size of the column is very small, This allows the polymer molecules (pectin) to be eluted separately from the lower molecular weight components such as salts and sugars. The effluent from the chromatography column was analyzed by measuring the refractive index (RI), normal and low angle laser light. It passes through four detectors: scattering (RALLS / LALLS), viscosity detector. Intrinsic viscosity is The concentration is calculated from the output of the viscometer detector in combination with a refractive index detector. The RALLS was used for dextran (control) and the refractive index detector In combination with this, Mw is calculated.

[0073] (Eluent) The eluent or solvent is a 0.3M lithium acetate buffer at about pH 4.6.

[0074] (Processing Procedure) (Standards and Controls) As a control standard, dextran with a molecular weight of about 64,000 daltons (concentration about 2.0 mg / mL) was used. As a control sample (working batch), pectin of known type IV (concentration 1 mg / mL) was used. Use L).

[0075] (Sample preparation) Manual sample preparation of milled pectin powder: (pectin and working batches) 1. Weigh out approximately 40.0 mg of sample into a 100 mL blue-capped bottle and add it to a semi-microcentrifuge. b) Use an analytical balance (readability limit 0.01 mg) to record the exact sample amount. 2. Add 100 μL of ethanol and drop the magnetic stir bar into the blue cap bottle. . 3. Add 40.0 mL of eluent and close the bottle with the cap. 4. Place the blue cap bottle on a magnetic stirrer. 5. Place the blue cap bottle in a storage heater at 75±2℃ and stir magnetically for 30 minutes. 6. After 30 minutes, place the bottle in a 5°C water bath for 5 minutes to cool to room temperature. 7. Transfer to autosampler vial.

[0076] (Analysis conditions) Eluent: 0.3M Li-acetate buffer pH 4.6. Flow rate: 1.0mL / min Pectin concentration: 1.0mg / mL Temperature: 37℃

[0077] (Protocol for measuring DM) (Procedure - Measure %DM only - Use indicator or pH meter / autoburette do) (Acid alcohol: 100mL 60% IPA + 5mL fuming HCl 37%) 1. Weigh 2.00 g of pectin into a 250 mL glass beaker. 2. Add 100 mL of acid alcohol and stir using a magnetic stirrer for 10 minutes. 3. Filter through a Büchner funnel lined with filter paper. 4. Rinse the beaker with 90 mL of acid alcohol. 5. Wash with 1000mL of 60% IPA. 6. Wash with approximately 30 mL of 100% IPA. 7. Dry the sample on the Buchner funnel with vacuum for approximately 15 minutes. 8. Weigh out approximately 0.40 g of sample into a 250 mL glass beaker. 10. Weigh out two samples for duplicate measurements. The deviation between the duplicate measurements should be greater than 1.5% absolute. otherwise the test must be repeated. 11. Moisten the pectin with approximately 2 mL of 100% IPA and mix using a magnetic stirrer. Add approximately 100 mL of deionized water.

[0078] This allows you to measure the pectin concentration of the sample using indicators or a pH meter / autoburet. Now we are ready to perform the titration (to calculate the deesterification rate of the carboxyl groups in the amine). This is explained below.

[0079] (Titration with indicator) 1. Add 5 drops of phenolphthalein indicator and drip with 0.1M NaOH until the color changes. Determine the titer (record this as V1 titer). 2. Add 20.0 mL of 0.5 M NaOH while stirring. Keep covered with foil. Leave it for 15 minutes. 3. Add 20.0 mL of 0.5 M HCl while stirring until the color disappears. . 4. Add 3 drops of phenolphthalein indicator and drip until the color changes with 0.1M NaOH. Determine the titer (record this as the V2 titer).

[0080] The first titration (V1) is used to quantify all carboxylic acid groups that are not methyl esterified. Next, a strong alkali is added to saponify the methyl ester, generating a free carboxyl instead. Then, in a second titration, these new carboxylic acid groups, which were the original ester groups, are Quantify (V2).

[0081] (manual calculation) Vt = V1 + (V2 - B1) (V2-B1) x 100 %DE (degree of esterification) = Vt %DFA (free acidity) = 100 - %DE 194.1xVtxCx100 %GA*(galacturonic acidity)=m *On an ashless and dry basis 194.1: Molecular weight of GA C: The corrected molar concentration of 0.1M NaOH used in the titration (e.g. 0.1002M) m: weight of the washed, dried sample for titration in mg Acid washed, dried pectin amount x 100 % pure pectin = weighed amount of pectin

[0082] Example 1: Extraction of pectin from dried lemon peel 1) Add 529.4 g of Na3-citric acid, 2·H2O to 36 L of deionized water and dissolve. Stir until combined. 2) Heat the solution to 60°C. 3) Add 900g of dried peel. 4) Incubate the mixture at 60°C for 150 minutes with gentle agitation. It should be sufficient to keep the hide pieces suspended and in motion. 5) Liquid is replaced with Dicalite 4258 (DICALITE EUROPE nv, B- 9000 GENT, http: / / www.dicalite-europe.com The retained material is discarded and the liquid passing through the bed is used for the following operations. Weigh and collect. (This liquid contains little or no insoluble material.) It is a solution without 6) Ion exchange resin Amberlite (trademark) SR1L NA Resin 25LT Bag, product number 10026751 (The Dow Chemical Company ny) is added in an amount of 50 mL per liter of liquid extract; The small beads are kept suspended by stirring for 30 minutes at ambient temperature. 7) The liquid extract was passed through a 60 μm nylon cloth (SEFAR PETEX 07-59 / 33 , Sefar AG, 9410 Heiden, Switzerland, filtra The retained material, i.e., the spent ion exchange The exchange resin is discarded. 8) Pour the fluid into 3 parts of 80% 2-propanol while gently stirring the 2-propanol. A fibrous precipitate of pectin is formed. 9) Pour the mixture onto a 60μm nylon cloth (SEFAR PETEX 07-59 / 33). The fabric is then folded around the retained solid material (fibrous precipitate) and squeezed by hand. The solid material is collected for the next operation. The liquid that passes through is discarded. 10) Dry the above solid material at 65°C for 10±2 hours. 11) The dried material is weighed and ground; the grinder is equipped with a 300 μm sieve. The powder that passes through this sieve is considered the pectin product.

[0083] Calculation of extraction yield: The pectin obtained in point (10) above weighed 76. The weight of the liquid after sieving was 13,375 g. The pectin yield per liter of extract was 5.74‰w / w. A total of 36 liters of water was used for the mixture. Pectin was extracted from 900 g of fruit peel. See point (3). Yield values ​​reported in this document are calculated as follows: Yield=(5.74·36 / 900)·100%w / w=23.0%w / w

[0084] Wherever yields are mentioned in this document, they are calculated in this way. Note that this is a calculation: the dry matter content of the peel as well as the dry pectin may vary slightly from 100%. The yield represents the amount of pectin dissolved under the extraction conditions, which includes The amount of pectin that would be lost in actual production is also included, because the above procedure Pectin becomes entrapped in the solid material discarded in (6) and (7), thereby This is because it cannot be extracted from the entire pectin-containing liquid.

[0085] Pectin analysis: IV=8.3 dl / g; DM=8.7.

[0086] Discussion of Results: IV shows that for citrus pectins with a DM of 8.7 or less, The yields were also surprisingly good, higher than any other known IVs using conventional extraction methods. This is comparable to what was previously considered a good yield.

[0087] Example 2, Extraction of Multiple Peel Lots with Na3-Citrate Several pectin samples were prepared under the manufacturing parameters specified in Table 1 below, otherwise It was prepared according to the same procedure as in Example 1. The yield, IV and DM obtained are shown in Table 1 below. PVIV is also quoted. PVIV is the yield multiplied by IV. These results Shown in Figure 2.

[0088] [Table 1-1]

[0089] [Table 1-2]

[0090] Discussion of Results: The method achieved consistent results across a range of peel lots with DMs below 30 and IVs below 5d. It has been shown to provide more than 1 l / g pectin. It has been shown to function in buffer solutions and at temperatures ranging from 60 to 74°C.

[0091] The pH conditions for the above samples were as follows: [Table 2]

[0092] Example 3: Extraction with other buffer salts Salts other than Na3-citrate can also be used for extraction. When using Na2H-citrate The pH during extraction is lower than when using Na3-citric acid. Table 2 shows extractions using other salts but otherwise following the same procedure as in Example 1. All experiments shown in Table 2 were carried out in a volume of 18 L. did.

[0093] [Table 3]

[0094] Discussion of the results: As can be seen, the various phosphate and citrate buffers significantly reduced the amount of phosphate in the skin. Stabilize the deesterification enzyme and release pectin with DM of 30 or less and IV of 5 dl / g or more. Promotes discharge.

[0095] The pH of these solutions was as follows:

[0096] [Table 4]

[0097] Example 4: Extraction with high peel / water ratio Large amounts of citrate appear to increase extraction yields, but at a cost.

[0098] High peel / water ratio and high citrate / water ratio to improve citrate availability Two extractions were performed with 100% ethanol, followed by sieving, dilution and shortly thereafter addition of Dicalite 42 In this way, the amount of peel used is only the normal consumption amount. Thus, citrate was able to be present in high concentrations throughout most of the extraction. The results of these experiments are summarized below.

[0099] [Table 5]

[0100] Example 5: Comparative Example Using Known Extraction Method The peel lots used in the experiment were subjected to quality control of the peels before being used for commercial pectin production. The standard extraction was also carried out by the conventional method, the so-called "standard extraction". As described in the paper, the peel was suspended in acidic water at 70°C for 7 hours, and then filtered to separate the pectin. Before extraction, the mixture is diluted with enough nitric acid to bring the pH to 1.7 during the extraction process. The DM of pectin produced by standard extraction is usually about 67 ± 4. The peanut extracts produced by standard extraction of these peel lots used in the experiments described herein were The characteristics of the cutin samples are summarized in Table 4 below.

[0101] [Table 6]

[0102] The various examples in Table 4 above (standard extractions) are the same as the examples in Table 1 (claimed methods). The experiment was carried out on a peel lot. For example, samples EB-04 to EB-07 in Table 1 have the same peel as sample 19 in Table 4. As can be seen from the comparison of these samples, the claimed method pectins with IV = 15.6-29.5 and IV = 5.29-6.30 were produced; Standard extraction produced pectin with DM=64 and IV=4.7. For samples EB-12 to EB-17 in Table 1, the same peel lot as sample 23 in Table 4 was used. As can be seen from the comparison of these samples, the claimed method achieves a DM of 12. pectin with IV=5.79-6.76 and IV=8-26.5 was produced; whereas the standard extract produced pectin with DM=65 and IV=5.4. Finally, samples EB-24 to EB-29 in Table 1 have the same peel as sample 25 in Table 4. As can be seen from the comparison of these samples, the claimed method pectins with IVs of 4.2-14.6 and IVs of 4.4-10.4 were produced; whereas the standard The extraction produced pectin with DM=66 and IV=5.6.

[0103] The other samples in Tables 5 and 6 are commercially available pectins, which are usually derived from mixed peel lots. Therefore, it was not useful for comparison.

[0104] Nonamidated LM-pectin, also known as "LMC-pectin", is currently commercially available Table 5 shows the properties of the LMC of some lots. Note that the V values ​​are much lower than the IV values ​​of the pectin samples of the present invention. There are no data on the production yield of individual pectin lots, but the yield was approximately 24% w / w of LMC. This yield is assumed to be representative of the production The commercial product types in the table below indicate the names of the pectin classes available. There are.

[0105] [Table 7-1]

[0106] [Table 7-2]

[0107] [Table 7-3]

[0108] [Table 7-4]

[0109] DKPA200201033 and US20070621747 are pectin-resistant strains with high DM. How to enzymatically deesterify pectin with a DM of about 30-40? The samples prepared according to this description are shown in Table 6. Please refer to the following.

[0110] [Table 8]

[0111] All the samples in Tables 1 to 6 are plotted in Figure 2 with DM on the x-axis and IV on the y-axis. In the figure, Hoejgaard pectin (see solid box) is represented. Chin is made by the microbial pectinesterase enzyme.

[0112] As can be seen from the table above and FIG. 2, the samples prepared by the novel procedures described herein The sample is very distinct from the prior art samples in that it is in the upper left portion of the figure, i.e. They have a unique combination of low DM and high IV. The composition of the present invention, produced by the novel process described in The dotted line is shown below 30.

[0113] The same samples are plotted in Figure 3 with yield on the x-axis and IV on the y-axis. Thus, samples made by the novel procedures described herein generally have no prior de-esterification. It has a higher IV and a higher yield than methods such as chemically deesterified pectin. (i.e., made by alkaline saponification or acid hydrolysis) It is represented by an unfilled diamond (H).

[0114] Example 6. Use of novel pectin to suspend cake beads in a delicate gel The pectin samples of the present invention and (for reference) some others outside the claimed parameters The solution of pectin sample was dry mixed with 0.25 g of pectin and 5 g of sucrose, and then The powder mixture was dispersed in 500 g of pure deionized water at room temperature with stirring. Stirring was continued for 10 minutes.

[0115] Test system: Haake Mars, Thermo Scientific, CPS100120, I I rheometer (Thermo Fisher Scientific, Waltham, Massachusetts) 1000 VA, ... ● Measurement geometry FL40 (FL40 vane spindle) ● Temperature regulator DC30 set to 25℃ ●Gap 4.000mm

[0116] procedure: a) 150 ml of 0.05% w / w pectin solution is placed in a cup (diameter 44 The tube was inserted into a 120 mm tube (120 mm in diameter and 120 mm long). b) A vane tool (FL40 - diameter 40 mm) was inserted into the fluid. c) While stirring, a measurement procedure was started at a shear rate of 25 sec-1 for 1 min. d) At the beginning of this measurement procedure, the sample was acidified by adding 2 ml of a 10% citric acid solution.

[0117] After a mixing time of 1 min, the solution or gel was subjected to an applied stress of 1.0 Pa and a vibration frequency of 1 H The apparent viscosity (symbol η*) and apparent storage elasticity after 30 minutes were evaluated by a vibration test at z. The rate (symbol G') was read.

[0118] The results are summarized in Table 7 below.

[0119] [Table 9]

[0120] These results can be compared relative to each other, but using a vane spindle Therefore, they cannot be absolute measurements of η* and G'.

[0121] The apparent η* and G', which are the rheological results, are independent parameters of pectin D To illustrate how the solubility of pectin depends on M, pectin IV, and pectin pH, Table The data from 7 was analyzed using MODDE software. The "pectin pH" here refers to the pure pH of The pH value obtained when pectin is dissolved in pure deionized water at a concentration of 0.3% w / w is The MODDE coefficients are summarized in Figure 4. The various solutions used in Table 7 above It has been shown that IV is the most influential parameter.

[0122] The pectin solutions in Table 7 are also used to suspend insoluble material in solutions or gels. "Silver Pearl" (Dr. Oetker Danmark AS, Sydv estvej 15, 2600 Glostrup) was gently stirred into the liquid; The "Silver Pearl" is a pearl with a diameter of about 4 mm designed to decorate desserts or cakes. The results are shown in Figure 5. The formulation in Figure 5 was gently poured into a glass container (shown in Figure 5) and It is semi-liquid or weakly gelled in the sense that it floats when rotated / twisted, but the movement of the container When the force is stopped, the beads reverse their original direction of motion and cease to move; this is evidence of a gel. As is evident from Figure 5, the beads are suspended in pectin solutions with high IV. In the pectin solution with a low IV, the silver particles remain in the same state, but in the pectin solution with a low IV, they settle to the bottom. The suspending action shown in the "Sodium phosphate" test is similar to that seen in other foods, such as salad dressings. It can also result in the suspension of chairs and vegetable pieces.

Claims

1. a) a degree of methyl esterification of 30 or less; b) The intrinsic viscosity is 5 dl / g or more. Citrus pectin, characterized by:

2. The pectin is orange pectin, lemon pectin, lime pectin or grape pectin. The citrus pectin of claim 1 which is a fruit pectin.

3. The degree of methyl esterification is: a) 20 or less; b) 15 or less; or c) 10 or less.

3. The citrus pectin according to claim 1 or 2.

4. 2. The method according to claim 1, wherein the intrinsic viscosity is a) 6 dl / g or more; or b) 7 dl / g or more.

4. Citrus pectin according to any one of claims 3.

5. The degree of methyl esterification is 30 or less; and the intrinsic viscosity is 5 dl / g or more. A deesterification method for producing pectin, comprising the steps of: The method comprises incubating in a solution comprising: a) a buffer that maintains the pH of the solution containing the peel at a pH of 5 to 8; b) Calcium binding agents.

6. An enzyme present in citrus peels for de-esterifying pectin in said citrus peels Use of.

7. The deesterification reaction is carried out in a buffer solution that maintains a pH between 5 and 8 and a calcium-binding The use according to claim 6, comprising an agent.

8. 6. The method of claim 5, wherein the concentration of the buffer for the deesterification is 0.02M or more. or the use according to claim 7.

9. The temperature of the deesterification reaction is: a) 55°C to 75°C; or b) 59°C to 65°C. The method according to claims 5 and 8 or the use according to any one of claims 6 to 8.

10. Any one of claims 5 and 8 to 9, wherein the calcium binding agent of b) is also the buffer solution of a). A method according to any one of claims 7 to 9 or a use according to any one of claims 7 to 9.

11. The calcium binding buffer comprises: a) Na ２ H-citric acid; b) Na ３ - citric acid; c) Na ２ H-phosphate; and d) Na ３ -phosphate The method or use according to claim 10.

12. 10. The method of claim 5, wherein the calcium binding agent comprises cation exchange beads. The method according to any one of claims 7 to 9 or the use according to any one of claims 7 to 9.

13. 13. Any of claims 5 or 8 to 12, wherein the incubation period is 30 minutes or more.

13. The method according to claim 1.

14. The citrus peel is orange, lemon, grapefruit or lime peel. The method according to any one of claims 5 or 8 to 13, or any one of claims 6 to 13.

2. The use according to claim 1.

15. Pectin obtainable by the method according to any one of claims 5 or 8 to 14.

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