Resistant starch and preparing method thereof

Abstract

The present invention relates to a method for preparing esterified flour as resistant starch, through cross-linking by esterification process through hot air oven and alternative method (reactive extrusion), comprising the steps of: for hot air oven, preparing acid aqueous solution; adding flour into the solution; incubating, and drying the slurry in hot air oven to get esterified flour; and washing the flour, and the resistant starch processed by the method thereof and for reactive extrusion, preparing and mixing flour with citric acid aqueous solution; reactively extruding the dried mixed flour; feeding the extruded dried mixed flour; adjusting a screw rotation speed of the extruding machine; stabilizing the weight of the starch citrate extrudate; grounding and sieving the stabilized starch citrate extrudate; and removing unreacted citric acid, and the neutral acid-base resistant starch citrate product processed by the method thereof. The purpose of the invention is to produce resistant starch citrate classified as resistant starch type 4 (RS4) to improve the digestion-resistant property of flour to be the key ingredient of the food helping develop the world food products, in the manufacturing scale, to have properties beneficial to health, as well as to add value to plant- based flour products.

Description

[0001] RESISTANT STARCH AND PREPARING METHOD THEREOF

[0002] Field of invention

[0003] The present invention relates to the field of manufacturing, biochemical engineering, food processing technology, and health impact, especially, relates to processes for preparing resistant starch, classified as resistant starch type 4 through cross-linking by esterification for improving resistant properties of natural flour and scientific proof of functional use as prebiotics.

[0004] Background of the invention

[0005] In the present, flour is powdery materials which mainly composed of starch with small amount of non-starch such as sugar, protein, lipid, and inorganic materials. Generally, starches consist of 20-30% amylose and 70-80% amylopectin. The process of making flour is by milling, grinding, and sifting the starch-containing plant organelles such as those from grains (rice (Oryza sativa var. indica), glutinous rice (Oryza sativa var. glutinosa), wheat, corn, rye, sorghum, and barley), seeds (almond, soy, and bean), roots (arrowroot, and yam), tubers (potato, and cassava), and fruits (green banana (GB; Hom Thong (Musa acuminata), Namwah (Musa balibisiana), and Hin (Musa sapientum Linn)). For a staple food in most cultures, cereal flour is one of the main ingredients in everyday meals. Since there is a high proportion of starch in the flour but still contains higher protein contents making flour harder and stronger, ensuring crusty and chewy breads whereas lower protein content will produce softer flour, which is perfect for noodles, cakes, and cookies. The ongoing studies and challenges in developing food products are to make modified starch with high resistant starch (RS) content in order to prevent and control of human diseases, especially colon cancer, diabetes, and obesity. Different methodologies can be used to increase RS formation and content in functional food applications.

[0006] RS is considered an insoluble fiber that is not hydrolyzed by digestive enzymes and enters the large intestine intact where it is partially or wholly fermented. RS can be classified into 5 types RSI, RS2, RS3, RS4, and RS5. RSI is physically inaccessible starch by nature such as unmodified cereals, seeds, and legumes. RS2 is a granular starch that strongly packed amylose structure that forms a crystallized molecule that can be found in green bananas, raw potatoes, high-amylose corn, and legumes. RS3 is called retrograded amylose or high amylose starches that can occur naturally during normal food processing, for example, cooked and cooled potatoes. Reheating and cooling with several cycles increase the amount of RS3. RS4 is a chemically modified RS that is not naturally occurring in foods. RS 5 is heat-stable and formed when lipids bind to amylose in the starch granule to prevent expansion of the granule, which is necessary for digestive enzyme hydrolysis. RS2, RS3, and RS4 can be added or formulated with other food supplements to work as a functional ingredient.

[0007] Generally, high dietary fibers in food are recommended for diabetic patients. To ensure that consistent amount of RS4 in flour production and derived products containing RS4, the procedure of starch citrate production has to manage alterations in the multi-scale structures of starch as the main composition in flour. The modification of starch granules and molecules breaks the original order and rearranges. The change in crystallinity and cross-linking reactivity via esterification leads to the better ability of gut microbiota to exploit RS. The gain benefits were closely linked to the modified structures or properties of RS (for example the crystallinity, molecular sizes, esterifying bonds, long or short chains, and surface particle morphology), which suggests that the competitive advantage of the strain depends on the fine structures of RS and would further result in a different response in the gut. The modifications may serve as a goal for the progress of RS as a functional and novel food in the future.

[0008] The method of manufacturing resistant starch comprises the step of selecting organic acid and organic base through a chemical reaction with flour. Organic acids play an important role in maintaining the nutritional value and sensory quality of foods and are also an important class of food additives, including their use as preservatives that affect bacteria, acidity regulators, and antioxidants. Organic compounds can be broadly classified in two manners: one is according to their structural characteristics and the other is based on their functional groups. Among organic compounds based on functional group which consists of carboxylic acid in the form carboxylic group -COOH in molecular structure. These are important functional groups for the appearance of esterification reactions in resistant starch crosslinking. Types of carboxylic acid are generally classified according to the differences in the number of carboxyl groups in the molecule; Monocarboxylic acid (formic acid, acetic acid, quinic acid) Dicarboxylic acid (Oxalic acid, succinic acid, malic acid, tartaric acid) and Tricarboxylic acid (Citric acid, Aconitic acid) (FIG. 1). All of those weak acids are Generally Recognized as Safe (GRAS) and have been approved by US Food and Drug Administration (FDA). The resistant starch from various organic used herein is considered a food product without causing deterioration in food flavor and maintaining organoleptic cooking quality. So, one or more of the organic acids on resistant starch manufacturing in food products described above can be provided not only as the explicit flavor but also as the physical properties of food products.

[0009] In consideration of preserving the sensory cooking characteristics, many sensory parameters are involved. Textural properties play a crucial role in determining eating quality and influencing consumer purchasing decisions for resistant starch products. The essential textural attributes for bakery products, namely hardness, chewiness, gumminess, adhesiveness, springiness, cohesiveness, and resilience are influenced by the raw material used. Hardness is the maximum force required to break the product, while chewiness refers to the physical characteristic of food that requires effort and time to break down and masticate while being eaten. Also, the food characteristic of gumminess refers to a specific textural quality in food characterized by a sticky or adhesive sensation that occurs when chewing. Moreover, adhesiveness refers to the sticking of surface food to different compositions such as tooth or mouth, it can be interpreted as a sticky mouthfeel. Furthermore, cohesiveness refers to the property of sticking within the food. Springiness is the height at which food can recover between the end of the first bite and the beginning of the second mouth. Finally, resilience is an indicator of how a food recovers from deformation.

[0010] Acid-heat treatment is a dry reaction method for the esterification of the starch to react with organic poly-carbonic acid under thermal treatment. This process induces the recrystallization of starch molecules without destroying the granule structure. This physical treatment involves incubating starch granules at a moisture content > 40% for a certain period at a temperature above the glass transition temperature but below gelatinization temperature. After the heating, the starch will be allowed to undergo cooling to facilitate the recrystallization of starch molecules. For alternative production processes, reactive extrusion (REX) is a simple process for chemically modifying of natural polymers, especially flour. In particular, twin-screw extruders are an excellent mixing and reacting device and can be used to produce modified flour with a more consistent quality in a continuous process. The REX process includes a high reaction temperature, high shearing forces, and chemical reagents that induce structural changes, by disrupting starch granules, melting crystallites, forcing molecular breakdown, and creating new helical structures. The melting or disruption of the initial crystalline structure of starch requires heating and shearing effects. The REX process yielding a more constant quality product with flexibility is from the design of the configuration of barrel sections and screw elements in a specific series of unit operations.

[0011] In the framework of the present invention, the RS content is the component of starch that is not hydrolyzed by digestive enzymes and passes through the intestinal tract with the rest of RS undergoing fermentation by resident microbiota. The formulation of dietary interventions with rich RS foods is proven to be associated with health benefits. For the determination, the method is analyzed according to the procedure of Englyst et al. (Classification and measurement of nutritionally important starch fractions, Europ. J. Clin. Nutr. (1992) 46 (Suppl.2), p33-p. 50). In the initial step of invention, flour is used as the starting raw material, specifically flour from GB- Hin, rice, and sorghum.

[0012] The consumption of RS containing foods provides the source of energy for the catabolism of intestinal microflora and the uptake of nutrients through the absorption of the intestine epithelial cells. Currently, various approaches have been employed to prepare RS including chemical, physical, and enzymatic modification methods. Chemical modification is one of the most well-known technologies for modified starch and RS preparation. Esterification is one of the processes that is mainly applied through chemical reactions resulting in the new bond energies for making a- 1,4 glycosidic bonds and a- 1,6 glycosidic bonds formed. This application increases the content of RS by the steric substitution of citric anhydride preventing the attack of amylase to digest and enter the large intestine intact, with the intestinal microflora fermentation by resident microbiota. Among microbiota, lactic acid bacteria (LAB) are widely used in the commercial production of fermented foods. These bacteria are classified as grampositive bacteria that exhibit excellent antibacterial activities because they can produce many antibacterial compounds such as organic acids, fatty acids, bacteriocins, cyclic dipeptides, and phenolic compounds. These bacterial species are able to produce lactic acid as the main end- product of carbohydrate fermentation. Not only LAB bacteria, but also Bifidobacteria which are one of the most important probiotic bacteria used in the dietary supplement with a great beneficial benefit to human diet. These bacteria are characterized as classified as non-sporeforming anaerobic bacteria, gram-positive with rod-shaped species. Both lactic and acetic acid can be produced as the major end-products of carbohydrate metabolism. These types of microorganisms are tolerated in acidic environments. This intestinal microbiota contributes the anti-inflammation, anti-aging, and cholesterol reduction. RS which had undergone hydrothermal treatment resulted in a greater increase in Lactobacilli and Bifidobacteria compared to untreated RS. Most results of the oxidative degradation of beneficial probiotic bacteria yield short-chain fatty acids (SCFAs) mainly acetic, butyric, and propionic acids. These SCFAs are essential for the well-being of human health, including maintenance of intestinal barrier integrity, regulation of the immune system, benefits for glucose and protein metabolism in the liver, protection against inflammation, reduction of the risk of colorectal cancer risk, control the appetite and combat against opportunistic bacteria. Not only, SCFAs play a very important role in preventing colon disease but also increase the absorption of minerals such as calcium, iron, and magnesium. These compounds seem to exert significant biological influences, such as diminishing early stages of colon cancer, regulating overall macronutrient processing, and modifying hormone release. These effects could contribute to enhanced physical and mental well-being. Several methods have been reported for producing various types of RS. These include US 5,281,276 issued on January 25, 1994, which described a process for making amylase RS from high amylose starch with the steps of gelatinizing a slurry of a starch and treating it with a debranching enzyme. After drying, extrusion or crystallization by the addition of salt was applied to produce the RS. Presently, esterification with citric acid is one of the most extensively used chemical modification methods for producing RS4 because of its high safety and thermal stability.

[0013] Hot air oven

[0014] Furthermore, US patent 11453730 B2 disclosing the method employs citric acid as the modifier, combines the citric acid with starch, achieves the good modification effect of starch at high temperatures, and improves the digestion-resistant property of starch. Citric acid itself is nontoxic, the process conditions are mild, and the treatment methods are relatively safe, which shows very high practical values. In other words, the US patent disclosed a method of improving the digestion properties of natural starch by mixing cereal starch with a citric acid solution. However, in conclusion, there is no invention similar to this invention focusing on the digestionresistant property improvement of flour.

[0015] Reactive Extrusion

[0016] US patent 11453730 B2 disclosing the method employs citric acid as the modifier, combines the citric acid with starch, achieves the good modification effect of starch at high temperatures, and improves the digestion-resistant property of starch. Citric acid itself is nontoxic, the process conditions are mild, and the treatment methods are relatively safe, which shows very high practical values. In other words, the US patent disclosed a method of improving the digestion properties of natural starch by mixing cereal starch with a citric acid solution. However, in conclusion, there is no invention similar to this invention focusing on the digestionresistant property improvement of flour.

[0017] W02021017231A1 discloses a method for making rice flour with a low glycemic index is disclosed. The invention involves adding a debranching enzyme and an emulsifier to rice starch, making a resistant starch through one-step extrusion, compounding other food raw materials, and then putting both through another extrusion to create the rice flour.

[0018] Furthermore, CN110372802B disclosing the method of making citrate starch described in the invention includes the following steps: mixing starch and citric acid; adding deionized water and an alkaline catalyst; uniformly mixing; sealing; placing in subcritical extrusion equipment; drying; crushing; sieving; washing with alcohol; and removing impurities to produce citrate starch with a substitution degree of at least 0.01. The method of making citrate starch by subcritical water extrusion has the benefits of achieving a substitution value of more than 0.01, being simple to use, allowing for continuous preparation, resolving the technical issue with the current method of making citrate starch, greatly advancing the industrial production of citrate starch, and having a large market.

[0019] Summary of the invention

[0020] This invention is involved in a method for preparing resistant starch citrate, through cross-linking by esterification process, comprising the steps of: preparing citric acid aqueous solution; adding flour into the solution; incubating, and drying the slurry in hot air oven to get esterified flour; and washing the flour, and the resistant starch citrate processed by the method thereof.

[0021] The purpose of the invention is to produce resistant starch citrate classified as resistant starch type 4 (RS4) to improve the digestion-resistant property of flour and to formulate the substituting refined flour with RS4 resulting in notable decreases in both blood glucose and reducing the risk for insulin resistance. This RS4 flour invention is to be the key ingredient of the food helping develop the world food products, on the manufacturing scale, to have properties beneficial to health, as well as to add value to plant-based flour products.

[0022] Brief description of the drawing

[0023] The invention is herein described, by way of example only, with reference to the accompanying drawings.

[0024] FIG 1A. illustrates the types of organic acid compounds based on the number of carboxylic groups: monocarboxylic, di carboxylic, and tricarboxylic.

[0025] FIG IB. illustrates the esterified starch from acid crosslinking of succinic acid, malic acid, tartaric acid, and citric acid.

[0026] FIG 2. illustrates an example of FUR spectrum of native and RS4 from GB-Hin, GB- Hom Thong, GB-Namwah, rice, sorghum, wheat, tapioca, and glutinous flour.

[0027] FIG 3. illustrates an example of FTIR spectrum of native and RS4 from GB-Hin flour, GB-Hin flour citrate, GB-Hin flour succinate, GB-Hin flour maleate, and GB-Hin flour tartrate that is prepared in accordance with the descriptions in Example 3 method for preparing a resistant starch with various type of carboxylic acid group.

[0028] FIG 4. illustrates an example of FTIR spectra of flour citrate using the continuous process of co-rotating twin reactive extruder with the reaction with 20% citric acid as native rice flour (green) and flour citrate at barrel zone 4 at 100°C (blue), 120°C (red), and 150°C (purple).

[0029] FIG 5. illustrates a gel permeation chromatography diagram of de-granular native flour (SWD-1-4) and RS4 flour citrate (SWD-5-8) of GB-Hin, GB-Hom, rice, and sorghum dissolved in DMSO by autoclaving at 120°C for 60 min.

[0030] FIG 6. illustrates different X-ray diffraction (XRD) pattern for two types of GB-Hin (native and RS4 flour citrate).

[0031] FIG 7A. illustrates different X-ray diffraction (XRD) pattern for two types of GB-Hom Thong (native and RS4 flour citrate) heat treatment by air drying.

[0032] FIG 7B. illustrates different X-ray diffraction (XRD) pattern for two types of GB-Hom Thong (native and RS4 flour citrate) heat treatment by freeze-drying. FIG 8. illustrates different X-ray diffraction (XRD) pattern of for two types of rice (native and RS4 flour citrate)

[0033] FIG 9. illustrates different X-ray diffraction (XRD) pattern for two types of sorghum (native and RS4 flour citrate)

[0034] FIG 10A. illustrates an example of changes in the morphology of native GB-Hin flour.

[0035] FIG 10B. illustrates an example of changes in the morphology of RS4 from GB-Hin flour citrate that are prepared in accordance with the descriptions in Example 1 method for preparing a resistant starch.

[0036] FIG 11 A. illustrates an example of changes in the morphology of acid heat treatment of native GB-Hom Thong flour (air-dried).

[0037] FIG 1 IB. illustrates an example of changes in the morphology of acid heat treatment of RS4 from GB-Hom Thong flour citrate (air-dried) that are prepared in accordance with the descriptions in Example 1 method for preparing a resistant starch.

[0038] FIG 11C. illustrates an example of changes in the morphology of acid heat treatment of native GB-Hom Thong flour (freeze-dried).

[0039] FIG 11D. illustrates an example of changes in the morphology of acid-heat treatment of RS4 from GB-Hom Thong flour citrate (freeze-dried) that is prepared in accordance with the descriptions in Example 1 method for preparing a resistant starch.

[0040] FIG 12A. illustrates an example of changes in the morphology of native rice flour.

[0041] FIG 12B. illustrates an example of changes in the morphology of acid heat treatment of RS4 from rice flour citrate (air dried) that is prepared in accordance with the descriptions in Example 1 method for preparing a resistant starch.

[0042] FIG 12C. illustrates an example of changes in the morphology of reactive extrusion rice flour citrate that are prepared in accordance with the descriptions in Example 1 method for preparing a resistant starch.

[0043] FIG 13 A. illustrates an example of changes in the morphology of native sorghum flour. FIG 13B. illustrates an example of changes in the morphology of RS4 from sorghum flour citrate that are prepared in accordance with the descriptions in Example 1 method for preparing a resistant starch.

[0044] FIG 14A. illustrates the bacterial number of Lactobacillus paracasei MSMC39-1.

[0045] FIG 14B. illustrates the bacterial number of Bifidobacterium animalis MSMC83.

[0046] FIG 15 A. illustrates the effect of RS4 from GB-Hin flour on caco-2 cell viability assessed by MTT assay.

[0047] FIG 15B. illustrates the effect of RS4 from GB-Hom Thong flour on caco-2 cell viability assessed by MTT assay.

[0048] FIG 15C. illustrates the effect of RS4 from rice flour on caco-2 cell viability assessed by MTT assay.

[0049] FIG 16A. illustrates the reduction of blood glucose level during OGTT and area under the curve (AUC) after short-term RS4 from GB-Hin flour (Musa sapientum Linn) intake.

[0050] FIG 16B. illustrates the reduction of blood glucose level during OGTT and area under the curve (AUC) after short-term RS4 from GB-Hom Thong flour (Musa acuminata) intake.

[0051] FIG 16C. illustrates the reduction of blood glucose level during OGTT and area under the curve (AUC) after short-term RS4 from rice flour (Oryza sativa var. indica intake.

[0052] Detailed description of the invention

[0053] The inventions to be disclosed herein are described in detail with reference to the illustrations. However, this invention may be provided in different aspects and ought not to be limited to the disclosed figure. The figure has been made so that this disclosure is absolutely clear so that those who are skilled in the art can fully understand the figure. However, the thickness and scale of the figure may be exaggerated for clarity.

[0054] Hot air oven

[0055] The present invention describes the preparation of resistant starch by esterification through the process of producing esterified flour under the combination of flour and citric acid, succinic acid, malic acid, and tartaric acid using the acid heat treatment method. The making of esterified flour yields RS4 which remains a high content of RS composition in the variety of processed food products. For the preparation of esterified flour with acid heat treatment method, the flour used in this modified esterified flour includes GB-Namwah flour, GB-Hin flour, GB- Hom Thong flour, rice flour, glutinous rice flour, tapioca flour, arrowroot flour, mung bean flour, and wheat flour. The citric acid, succinic acid, malic acid, and tartaric acid solutions were prepared at a concentration of 10% to 25% (w / v) and adjusted to pH 2.0 to 3.5 with 10 Molality (M) of sodium hydroxide solution. The esterification of flour was homogenously dispersed into a slurry and then mixed with the acid solution at a ratio of flour: acid solution from 1 :1 to 1 :1.8, w / v, then incubated for 16 hours at room temperature and dried at 50°C. The dried flour was then esterified at 140-160°C for 3 to 6 hours in a hot air oven and washed with sodium bicarbonate followed by twice times of distilled water to remove unreacted acid until the final pH to 7.0. The drying of esterified flour was used after centrifugation to reduce the moisture content to 3% to 7% at 50°C for 24 hours. The esterified flour was kept in a closed container. For the measurement of RS4 formed, a determination of the resistant starch content can be performed as a resistant starch (RS) content. This method was measured using the procedure of an enzymatic assay kit from Megazyme (Wicklow, Ireland) for the measurement and analysis of resistant starch in plant materials and starch samples. This method was according to official analysis methods: AO AC Method 2002.02, AACC Method 32-40.01, CODEX Type n Method.

[0056] A method for preparing a resistant starch comprising the steps of:

[0057] (a) preparing monocarboxylic acid (formic acid, acetic acid, propanoic acid, and quinic acid), dicarboxylic acid (succinic acid, malic acid, tartaric acid, and oxalic acid), and tricarboxylic acid (citric acid and aconitic acid) aqueous solution with the concentration ranging from 10% to 50%, preferably ranging from 10% to 25%;

[0058] (b) adjusting the pH of the acid solution obtained from step (a) with 5 to 10 M of sodium hydroxide to the pH of 2.0 to 3.5;

[0059] (c) adding flour into the acid aqueous solution with the ratio of the flour and acid of 1:1 to 1: 1.8, w / v to have a mixture of the flour and the acid to get slurry, the preferred flour is selected from one of the following flour sources comprising freeze-dried and air-dried tapioca flour, and / or rice flour, and / or glutinous rice flour, and / or wheat flour, and / or GB-Hom Thong flour, and / or GB-Hin flour, and / or GB-Namwah flour, and / or sorghum flour, and / or mung bean flour, and / or arrowroot flour, or a combination thereof; (d) incubating the slurry obtained from the step (c) for a first period of time ranging 12-20 hours from at room temperature, preferably 16 hours;

[0060] (e) firstly, drying the incubated slurry obtained from the step (d), to reduce the moisture content of the flour to less than 5% at 50°C for a ranging time from 24 to 48 hours, preferably 24 hours;

[0061] (f) heating the dried flour obtained from the step (e), for a second period of time ranging from 3 to 6 hours, preferably 5 hours, in hot air oven at 140 to 160°C to get esterified flour;

[0062] (g) washing the esterified flour obtained from the step (f) with sodium bicarbonate and distilled water to remove unreacted acid, preferably, two times washing

[0063] (h) secondly, drying esterified flour obtained from the step (g) to reduce the moisture content of the flour to 3% to 7% at 50°C for a third period of time ranging from 24 to 48 hours, preferably at 50°C and 24 hours

[0064] Additionally, the method of preparing the resistant starch according to this invention further comprises a step of keeping the esterified flour obtained from the step (h) in a closed container.

[0065] The resistant starch product processed by the method according to this invention contains highly resistant content to enzymatic digestion in a range of 3% to 65%, remains resistant starch, as RS, ranging from 7% to 34%. Furthermore, the resistant starch product processed by the method according to this invention is able to be used as a prebiotic to boost the growth of gut microbes, Lactobacillus paracasei MSMC39-1 and Bifidobacterium animalis MSMC83.

[0066] Reactive extrusion method

[0067] The present invention describes the preparation of resistant starch by esterification through the reactive extrusion method. The making of starch citrate yields RS4 which remains a high content of RS composition in a variety of processed food products.

[0068] A method for preparing resistant starch through reactive extrusion comprising the following steps: (a) preparing and mixing 1.5 to 3.5 kilograms of flour with citric acid aqueous solution, wherein the concentration of the solution ranges from 10% to 40%, to get 10 to 20% moisture content of the dried mixed flour, the preferred flour is selected from one of the following flour sources comprising dried and air-dried tapioca flour, and / or rice flour, and / or glutinous rice flour, and / or wheat flour, and / or GB-Hom Thong flour, and / or GB-Hin flour, and / or GB-Namwah flour, and / or sorghum flour, and / or mung bean flour, and / or arrowroot or a combination thereof;

[0069] (b) reactively extruding the dried mixed flour obtained from the step (a) comprising: conveying via, mixing, and kneading within, and passing the flour through a heated barrel of extruding machine configured to have a least six heating zones to obtain extruded dried mixed flour, wherein a ratio of barrel length and barrel length diameter (L / D) ranges from 28 to 36: 18 to 26;

[0070] (c) feeding the extruded dried mixed flour obtained from the step (b) with the feeding rate ranging from 5 to 10 kilograms per hour (kg / h) at barrel input zones, wherein the feeding is away from a metering and die zones;

[0071] (d) adjusting the screw rotation speed of the extruding machine to range from 200 to 250 rpm with a torque of 40% with the temperature from 30 to 150°C in the barrel to get starch citrate extrudate;

[0072] (e) stabilizing the weight of the starch citrate extrudate, obtained from the step (d), to be constant comprising collecting, transferring in a hot air oven, and drying the starch citrate extrudate at 40 to 50°C;

[0073] (f) grounding and sieving the stabilized starch citrate extrudate obtained from the step (e) through 60 to 90-mesh sieves to get the sieved dried mixture;

[0074] (g) removing unreacted citric acid comprising: washing the sieved dried mixture obtained from the step (f) with absolute ethanol or water or 0.5 to 2 M of sodium bicarbonate to get pH 7 of resistant starch citrate product.

[0075] The weight of flour, the concentration of citric acid aqueous solution, and the moisture content of the dry mixed rice flour of the step (a) preferably ranging from 2 to 3 kilograms, ranging from 10% to 20%, and 14% to 20% respectively. Moreover, the extruding machine of the step (b) was selected from a single-screw extruder, a twin-screw-extruder, or a co-rotating twin-screw extruder, preferably, the co-rotating twin-screw extruder, wherein a barrel length and barrel length diameter ratio (L / D) is preferably 32:22. The feeding rate of the flour, and barrel input zone are preferably 8 kilograms per hour (kg / h), and a barrel input zone 4 respectively.

[0076] Furthermore, the rotation speed and torque of the step (d) are preferably 250 rpm and 47%, the temperature to stabilizing starch citrate extrudate of the step (e) is preferably 45°C, and the sieve of the step (f) is preferably 80-mesh sieve. The washing time with the absolute ethanol, water, and 1 M of sodium bicarbonate solution is preferably four times.

[0077] Additionally, the method of preparing the resistant starch citrate according to this invention further comprises a step of keeping the starch citrate obtained from the step (g) in a closed container.

[0078] The resistant starch citrate product processed by the method according to this invention contains high resistant content to enzymatic digestion in a range of 30% to 50%.

[0079] EXAMPLE 1

[0080] Effect of raw materials on the characteristic of esterified flour

[0081] The preparation of esterified flour with acid heat treatment method, the flour used in this modified esterified flour includes GB-Namwah flour, GB-Hin flour, GB-Hom Thong flour, rice flour, glutinous rice flour, tapioca flour, arrowroot flour, mung bean flour, and wheat flour. The citric acid solutions were prepared at a concentration of 10% to 20% (w / v) and adjusted to pH 3.5 with 10 M of sodium hydroxide solution. The esterification of flour was homogenously dispersed into a slurry and then mixed with the acid solution at a ratio of flour: acid solution from 1: 1 to 1:1.8, w / v, then incubated for 16 hours at room temperature and dried at 50°C. The dried flour was then esterified at 160°C for 5 hours in a hot air oven and washed with sodium bicarbonate followed by twice times of distilled water to remove unreacted acid until the final pH to 7.0. The drying of esterified flour was used after centrifugation to reduce the moisture content to 3% to 7% at 50°C for 12 hours. The esterified flour is kept in a closed container.

[0082] The esterified flour was prepared by hot air oven method as described. The characteristic of esterified flour was determined in terms of resistant starch content and non-resistant starch by Megazyme (Wicklow, Ireland) test kits. This method was according to official analysis methods: AO AC Method 2002.02, AACC Method 32-40.01, CODEX Type II Method. In brief, samples of finely milled (0.5 mm) flour and food samples were weighed into 16.5 x 101 mm, 13 ml polypropylene tubes, and the tubes were tapped gently to ensure that all samples fell to the bottom of the tube. In detail, the dried sample (200 mg) was treated with 8 mL of an enzyme mixture (a-amylase and amyloglucosidase) in a shaking water bath at 37°C for 16 hours. The undigested flour was washed three times with 50% ethanol. The residue was dissolved with 0.5 mL of 2 M KOH in a shaking ice bath for 20 min. Then the mixture was adjusted to pH 4.75 with acetate buffer (pH 3.8), and hydrolyzed into glucose by using amyloglucosidase (3300 U / mL) at 50°C for 30 min. The released glucose concentration was determined using a glucose oxidase / peroxidase reagent (GOPOD). In detail, aliquots (0.1 mL, in duplicate) of reacted flour samples were treated with 3.0 mL GOPOD reagent and incubated at 50°C for 20 min. Reagent blank solutions contained 0.1 mL 0.1M sodium acetate buffer, pH 4.5, and 3.0 mL GOPOD reagent. Glucose standards (prepared in quadruplicate) contained 0.1 mL glucose (1 mg / mL) and 3.0 mL GOPOD reagent. After incubation at 50°C for 20 min, the absorbance of each solution was measured at 510 nm against the reagent blank. The glucose content of the supernatant and digested pellet was used in the calculation of total starch (TS) and Resistant Starch (RS) respectively by applying the factor of 0.9.

[0083] Moreover, the esterified flour was determined by water holding capacity, swelling power, and solubility percentage. The flour samples (100 mg dry basis) were soaked in deionized water (10 mL). The solution was agitated at 25°C for 6 hours before centrifugation at 14,000xg for 15 minutes. Before evaporation of the sample at 105°C, the supernatant was carefully removed and weighed (g). Water holding capacity was calculated using a gram of adsorbed water per gram of dried material. Swelling power and solubility percentage were determined by mixing samples (0.4 g dry basis) with deionized water (40 mL) in a centrifuged tube and then heated for 30 minutes at a temperature range of 40 to 90°C with an interval of 10°C. The suspensions were then centrifuged for 10 minutes at 3000xg. The supernatant was decanted, and the sediments were weighed before being dried at 105°C. The swelling power was calculated as the weight of wet sediment / weight of the dried sample. The solubility was calculated as (weight of dissolved solids after evaporation / weight of original sample) xlOO. The esterified flour was determined particle size using a laser particle size analyzer (Mastersizer 3000, Malvern, United Kingdom). Furthermore, the starch size distribution of the esterified flour was determined by high-performance size extrusion chromatography. Twenty milligrams of esterified flour were dissolved in 1 mL of DMSO and autoclaved for 1 hour at 121°C before being diluted with water to a final concentration of 4 mg / mL and filtered through a 0.45 m nylon syringe filter. Twenty microlites of the sample were injected into a Shimadzu HPLC system consisting of a LC-20AD pump, RID-10A detector, three serially connected columns (Ultrahydrogel linear and two Ultrahydrogel 120 column, Waters, USA) with a specific guard column, and a computer with a data analysis software program (LabSolutions). An isocratic elution with water was carried out at 60°C and a flow rate of 0.8 mL / min.

[0084] The change in the functional groups present in the native and esterified starch citrate was analyzed using a Fourier transform infrared spectrometer (Invenio S, Bruker, United Kingdom). The ATR spectra for flour samples were analyzed by performing 32 scans / min. The spectrums were recorded from 4000 cm'1to 500 cm'1at a resolution of 2 cm'1. The samples were subjected to the FUR analysis.

[0085] The crystallinity of flour for the native and esterified solids was carried out in a powder X-ray diffractometer (Model: SmartLab X-Ray diffractometer, Make: Rigaku, USA). The analysis was carried out from 50 to 500 with Cu-Ka as the monochromatic radiation. The crystallinity index of the solid sample was calculated based on Segal's expression according to the following equation:

[0086] Crystallinity index (CrI), % = ((I002 -lam) / 1002) x 100 where I002 is the intensity of the crystalline region for cellulose identified at a Bragg's angle (20) of 22° and Iamis the intensity of the amorphous region for cellulose identified at a Bragg's angle (20) of 18°.

[0087] The morphology of native flour and citrate-esterified flour was studied by scanning electron microscopy (SEM; QUANTA 450, JEOL Ltd., Tokyo, Japan). The flour samples were fixed onto the surface of double-sided carbon-coated adhesive tape attached to an aluminum stub. The dried flour samples were run in a low vacuum Micrographs were taken for each sample at a magnification of 5000X. Pasting properties of the samples were tested using a Rapid ViscoAnalyzer (RVA 4, Newport Scientific, Australia). The analysis using a 4 g flour sample and 25 g distilled water was placed in an aluminum sample canister. The RVA pasting curve was obtained by using a 30- minute test profile; initial equilibrium at 30°C for 10 min, heating to 95°C over 6 min, holding at 95°C for 5 min, cooling to 50°C over 5 min, and holding at 50°C for 4 min. The peak viscosity, breakdown viscosity, and final viscosity values were evaluated with the data analysis software (Thermocline for Windows, Newport Scientific, Australia).

[0088] In a particularly preferred embodiment of the process according to the invention, the esterification of flour and acid solution, the swollen flour that is mainly composed of starch chains reacted with acid at high temperature, 160°C resulting in the reduction of the in vitro digestibility of esterified flour. Specifically, the establishment of the cross-linking structure by an esterification reaction of the hydroxyl group in the starch chain with the acid anhydride led to the high content of RS. Therefore, the change in the internal structure and physicochemical properties of the esterified flour occurred such as the water holding capacity, the degree of swelling, and the water solubility. The different raw materials had an effect RS content and the physical characteristics as illustrated in Table 1. At 20% wt. of citric acid concentration, the highest resistant starch content of esterified flour was prepared from GB-Hin flour.

[0089] TABLE 1

[0090] Resistant starch content and physical characteristics in terms of water holding capacity, swollen capacity, and solubility of esterified flour prepared from different raw materials.

[0091] Pasting profiles and viscosity parameters of native and citrate flours were tabulated in Table 2. The peak viscosity, breakdown, final viscosity, and setback of starch citrate significantly reduced when subjected to acid heat treatment using a convection oven because multiple esterification among starch molecules by citric acid may occur leading to the inhibition of their solubilization and swelling.

[0092] TABLE 2.

[0093] Pasting properties of native and esterified flour prepared from different raw materials.

[0094] *ND means not detected.

[0095] FIG. 2 illustrates the infrared spectra in the region of 4000-500 cm'1of native and rice, glutinous rice, GB-Hin, GB-Namwah, GB-Hom Thong, sorghum, and tapioca esterified flour. In the range of the FTIR spectrum around 1300-900 cm'1reflected the C-C, C-OH, and C-H stretching vibration. This region was sensitive to changes in starch conformation and the process of hydration. The band peaks at 999 and 1049 cm'1corresponded to the molecular order and crystallinity of starch polymers, their intensity increases as the crystallinity of starch increases. In native flour normally IR band in the range of 3100-3700 cm'1was assigned to OH group (O-H stretching). The lower absorbance showed the dehydration of water out of the molecular structure of starch during the mixing process with the high temperature. For the wavelength of 2800-3000 cm'1showed CH2 groups (C-H bond stretching) while around 2928 cm'1showed O-H groups and CH2 deformations. For 1642 cm' 1 reflected the carboxylate ion (COO) and 1500 showed skeletal mode vibration of alpha- 1,4 glycosidic linkages (C-O-C). In the range of 1300-1350 cm'1, this spectrum revealed C-O-H, C-C-H, and C-O-H bending. The band 1150 (1149.63) and 1080 (1077.10) cm'1can be interpreted as coupling of C-O, C-C, and O-H bond stretching bending and asymmetric stretching of the C-O-C glycosidic bridge. The 999.71 and 1077.10 cm'1showed molecular order and crystallinity region of starch polymer where both 929.75 and 860.85 cm'1illustrated C-H bending. Specifically, in the IR band at 860.85 cm'1of C-O-C symmetrical stretching and C-H deformation can be identified. For the region of 620-572 cm'1wavelength, the skeletal modes of the pyranose ring are revealed. For the esterified flour, the changes in structure on a molecular level such as starch chain conformation, crystallinity, and retrogradation can be detected throughout the region of 4000-500 cm'1. Compared with the native flour, a new peak of 1720-1737 cm-1appeared in all esterified flour samples. The new absorbance peak can be attributed to the characteristic ester group showing the stretching vibration of C=O bond from the citric acid in the structure of esterified flour.

[0096] According to particle size determination, a diagram showing the particle size distribution of resistant starch type 4 of GB-Hin, GB-Hom, rice, and super sorghum was shown in FIG. 3. In Table 3, D[4,3] represented weighted mean value by volume. The result showed that the esterified flour from sorghum had the highest D[4,3] value while the lowest D[4,3] was presented in the esterified flour from GB-Hin.

[0097] TABLE 3.

[0098] D [4,3] of esterified flour prepared from different raw materials. The starch size distribution of native and esterified flour is shown in FIG. 4. The results showed that after the esterification process, the proportion of high starch size of all esterified flour was reduced when compared to its native counterparts due to acid hydrolysis.

[0099] Differences in the crystallinity degree of flour can be observed by the XRD technique.

[0100] All the samples had clear A-type diffraction patterns, with main reflections approximately at 29 ~ 15°, 17°, 18° and 23°. Changes in the starch crystal structure can be caused by damage due to the temperature rise or the crystalline structure reorganization due to the loss of water. Therefore, the flour crystalline structure of these chemically modified samples was transformed from A-type to Vu-type by esterification and / or cross-linking. The relative crystallinity in the different starch citrate can be varied as shown in Table 4. TABLE 4.

[0101] The crystallinity index (CrI) of native and esterified flour prepared from different raw materials.

[0102] FIG. 10 to 13 showed the results from using SEM to analyze the granular morphology and surface structure of flour. The native flour granules of GB-Hin flour demonstrated a smooth surface and a shape of oval and ellipsoid granules. For rice flour, micro images showed polyhedral starch granules having irregular shapes, with sharp angles and edges, and without pores on the smooth surface. For sorghum flour, the rounded spherical and oval, and a few were irregular granules with different sizes. During the process of water absorption, expansion, and re-cooling of the native flour, the flour molecules can be rearranged to form a dense crystalline structure due to the influence of internal and external factors as shown in FIG. 10B, 11B, 11D, 12B, 12C, and 13B resulting in the rearrangement to form new crystalline structures.

[0103] EXAMPLE 2

[0104] Effect of temperature, reaction time, and percentage of acid concentrations during the esterification process on resistant starch content of esterified flour.

[0105] In this example, the esterified flour was prepared by the hot air oven method as described in Example 1 with different conditions. The preparation of esterified flour with acid heat treatment method, flour used in this modified esterified flour includes GB-Hin flour, rice flour, tapioca flour, and wheat flour. The citric acid solutions were prepared at a concentration of 10% to 20% (w / v) and adjusted to pH 3.5 with 10 M of sodium hydroxide solution. The esterification of flour is homogenously dispersed into a slurry and then mixed with the acid solution at a ratio of flour: acid solution from 1 :1 to 1 : 1.8, w / v, then incubated for 16 hours at room temperature and dried at 50°C. The dried flour is then esterified at 140 to 160°C for 3 to 6 hours in a hot air oven and washed until the final pH to 7.0. The drying of esterified flour is used after centrifugation to reduce the moisture content to 3% to 7% at 50°C for 12 hours. The resistant starch (RS) and non-resistant starch (non-RS) contents of esterified flour were determined as described in Example 1. Moreover, total starch (TS) content and percentage of resistant starch to total starch (RS / TS) were calculated.

[0106] From Table 5, the RS and RS / TS of esterified flour prepared at 160°C were higher than those of esterified flour prepared at 140°C, while the non-resistant starch content of esterified flour prepared at 160°C was lower. For the esterification time, the optimum time for preparing acid-esterified flour was 5 hours, the result is shown in Table 6. However, the high acid concentration resulted in increasing RS and RS / TS (Table 7). These are due to the low moisture content and increase in acid anhydride at higher temperatures. TABLE 5.

[0107] Effect of esterification temperature on resistant starch content.

[0108] TABLE 6.

[0109] Effect of esterification time on resistant starch content.

[0110] TABLE 7.

[0111] Effect of acid concentration on resistant starch content. EXAMPLE 3

[0112] Effect of type of acid on the characteristic of esterified flour.

[0113] Types of carboxylic acid are generally classified according to the differences in the number of carboxyl groups in the molecule; monocarboxylic acid (formic acid, acetic acid, and quinic acid) dicarboxylic acid (oxalic acid, succinic acid, malic acid, and tartaric acid) and tricarboxylic acid (citric acid, and aconitic acid). The resistant starch from various organic that used herein are considered as food products without causing deterioration in the flavor of food. Types of acid that are used for preparing esterified flour influenced resistant starch content. In this example, the different types of acid including citric acid, succinic acid, malic acid, and tartaric acid were used for preparing esterified flour as described in Example 1. The citric acid, succinic acid, malic acid, and tartaric acid solutions were prepared at a concentration of 10% to 25% (w / v) and adjusted to pH 2.0 to 3.5 with 10 M of sodium hydroxide solution. The esterification of flour is homogenously dispersed into a slurry and then mixed with the acid solution at a ratio of flour: acid solution of 1 :1.8, w / v, then incubated for 16 hours at room temperature and dried at 50°C. The dried flour is then esterified at 160°C for 5 hours in a hot air oven and washed until the final pH to 7.0. The drying of esterified flour is used after centrifugation to reduce the moisture content to 3% to 7% at 50°C for 12 hours. The characteristics of esterified flour prepared from different types of acid are shown in Table 8. The results presented that esterified flour prepared from citric acid had the highest resistant starch content. For FTIR analysis, a new peak of all esterified flour from citric acid, succinic acid, malic acid, and tartaric acid appeared at 1730 to 1750 cm-1as described in example 1 (FIG. 4). The new peak at 1730 to 1750 cm'1was ascribed to the carboxyl and ester carbonyl bands. It suggested that all acids that were used in this example can prepare the esterified flour by crosslinking with acid.

[0114] TABLE 8.

[0115] Effect of type of acid for esterification process on resistant starch content and physical characteristics.

[0116] EXAMPLE 4

[0117] Alternative method for preparing esterified flour.

[0118] Rice flour was selected to prepare esterified flour by reactive extrusion (REX). A corotating twin-screw extruder (Chareontut Co., Ltd, CTE-D22L32, Thailand) was used for the REX process. The extruder has a barrel length to barrel length diameter ratio of 32:22. The process of loading 14% moisture content of the dry mixed rice flour with 20% citric acid was conveyed, mixed, kneaded, and passed through a heated barrel journey with six heating zones under the varying temperatures at barrel zone 4 for reactivity without the metering and die zone at a feed rate of 8 kg / h. After the operation was prepared, the flour was loaded to were run through the six barrels at adjusted temperatures accordingly; Condition 1 : 30, 80, 80, 100, 90, 75°C; Condition 2: 30,100, 100, 120, 110, 100°C and Condition 3: 30, 130, 130, 150, 140, 130°C at the screw speed of 250 rpm with torque 47%, respectively. The starch citrate extrudate samples were then collected, transferred in an oven, and dried to constant weight at 45°C before ground and sieved through an 80-mesh sieve. The dry mixture was washed with absolute ethanol four times and finally with 1 M sodium bicarbonate to get rid of the unreacted citric acid and pH reached 7. Table 9 shows the resistant starch content under different conditions to modify by co-rotating reactive extrusion.

[0119] TABLE 9.

[0120] Resistant starch content passed through a twin screw extrusion process. a,bMean value and standard derivation of five measurements The FTIR spectra of the non-cross-linked native rice flour and extruded crosslinked flour with 20% citric acid were shown in FIG. 4. The amplitude of extruded flour RS in the transmission bands between 3100 and 3700 cm'1which corresponds to the -OH group was also found to be different from that of native rice flour, indicating the changes in the combination of hydrogen bonds generated during the formation of RS. For chemical- modified RS4, it was noticed that the appearance of transmittance at wavenumber 1716 cm-1. The band at 1716 cm-1(indicated by an arrow in FIG. 4) was ascribed to the carboxyl and ester carbonyl bands. The changes in the amplitude in transmission bands on FTIR suggested that the appropriate condition in the twin screw can prepare the rice flour to create the crosslinking of starch citrate. The more pronounced peaks at 1716 cm'1confirmed the esterification of crosslinking occurred in all treated temperatures (100, 200, and 150°C).

[0121] The relative crystallinity in the starch citrate by reactive extrusion is shown in Table 10. The results showed that the esterified flour prepared by acid-heat had lower relative crystallinity than the esterified flour prepared by reactive extrusion due to the mechanical or shearing force effect. However, the flour crystalline structure of these chemically modified samples was transformed from A-type to Vu-type by esterification and / or cross-linking.

[0122] TABLE 10.

[0123] The crystallinity index (CrI) of esterified flour is prepared by different methods.

[0124] For reactive extrusion rice flour, the shape and form of rice granules showed extreme damage than acid-heat treatment flour (FIG. 12C). When rice flour was heated in a series of heated barrels with the presence of a mechanical or shearing force, it changes its granular structure, such as granular disruption, melting of the crystalline zones, and the formation of a three-dimensional network; hence, the more fracture zone of the material was observed. However, reactive extrusion is one of the alternative methods for preparing resistant starch.

[0125] EXAMPLE 5 Characteristics of esterified flour products.

[0126] Chiffon cake was selected to determine the effect of esterified flour prepared from different acid cross-linking conditions on products. After baking, the color of the chiffon cakes was measured by a colorimeter. The texture of chiffon cakes was evaluated by a texture analyzer (TAXT Plus, TA Instruments, United Kingdom). The texture properties were performed when cake crumb samples in the form of cubes measuring 2 cm sides were cut from the center of the cake. The probe used to compress the sample was P / 100. The texture profile analysis (TP A) was performed at 1 mm / s pre-test speed, 1 mm / s test speed, and 10 mm / s post-test speed with 30 seconds holding time between the compressions. First, the cake was compressed by the probe up to 75% of its height before returning to its beginning position for 30 seconds. Then, the sample was compressed again by the same strain level. Compression force, elapsed time, and areas under the compression force were measured. Hardness, chewiness, gumminess, adhesiveness, springiness, cohesiveness, and resilience values were calculated, twenty readings were carried out from different samples of the same set. The results are shown in Table 11.

[0127] TABLE 11.

[0128] The textural properties of chiffon cake prepared from esterified flours.

[0129] Color measurement (L*, a*, and b* values) of chiffon cake (crust and crumb) samples was performed using a portable colorimeter (3nh model NH 300). L* (Lightness) indicates lightness intensity from 0 (black) to 100 (white). The a* indicates color intensity from greenness (negative values) to redness (positive values) while b* indicates color intensity from blueness (negative values) and yellowness (positive values). The result illustrated that the highest L* value of chiffon cake crust was chiffon cake prepared with esterified flour with succinic acid (Table 12). The highest L* value of chiffon cake crumb was chiffon cake prepared by esterified flour with malic acid. TABLE 12.

[0130] Color of chiffon cake prepared from esterified flours.

[0131] Variation in RS Content in Foods

[0132] The variation in RS content within and between each food category can be influenced by the composition of the formulated food and the method of cooking. The remaining RS contents of some type of food after processing were quantified and reported as Table 13.

[0133] TABLE 13.

[0134] Effects of processing and physicochemical properties of esterified flour as RS4 in the invention foods.

[0135] Any change made to this invention may be vividly understood and can be done by a person skilled in the field. The change may be within the scope and intent of this invention as shown in the claim attached.

[0136] EXAMPLE 6 Health impact of esterified flour

[0137] Viability of the probiotic microorganisms

[0138] The cell viability of the probiotics was performed by incubating the starch citrate with two strains of probiotics as Lactobacillus paracasei MSMC39-1 and Bifidobacterium animalis MSMC83 at 108CFU / mL in anaerobic jar at 37°C for 24 hours After the incubation period, the count of viable probiotic cells was carried out using plate count in De Man, Rogosa and Sharpe agar with the serial dilution from 10°-10'12.

[0139] The result was expressed as colony-forming units per milliliter. Results showed that the number of L. paracasei MSMC39-1 of GB-Hin starch citrate increased from 7xl08to 6.3xl09. The four times increment (from 2.6xl08to 8.3xl08) of B. animalis MSMC83 was detected in the cell viability (FIG. 14A and 14B). Prebiotic index from the fluorescence in situ hybridization

[0140] Fluorescence in situ hybridization (FISH) presents a promising path for rapidly and accurately measuring bacterial quantities in probiotic products. FISH can illustrate bacterial growth trends in the real-world contexts of product application, eliminating the requirement for supplementary growth restrictions that are obligatory when using plate counts for quantification. Fluorescent In Situ Hybridization (FISH) tests the growth of bacteria in the intestines under conditions with resistant starch compared to the non-supplemented group (control) and the group supplemented with 1% glucose. The sample product, comprising 1% of the local fecal bacteria, was combined with feces by using 2.5 g of feces dissolved in 5 mL of Nutrient Broth. This mixture was homogenized and then centrifuged at 4,000 rpm for 10 minutes to separate fecal particles. Next, the separated liquid portion was diluted in a feeding medium at a ratio of 50 mL per 1 mL of medium. Then, the sample products were incubated at 37°C for 24 hours. Subsequently, the bacteria in the sample before and after were kept in 4% formalin solution at 4°C for 24 hours. After that, the sample was washed with phosphate- buffered saline (PBS) by centrifuging at 6,500 rpm for 10 minutes, repeating this process twice. Then, the sample was stained with 4',6-diamidino-2-phenylindole (DAPI) to enumerate the total bacterial count. Next, stain with specific FITC-labeled probes for Lactobacillus (Lac), Bacteroides (Bac), Bifidobacterium (Bif), and Clostridium (Clos) according to Table XX to quantify the bacterial counts of each aforementioned group. The samples stained with fluorescent agents were counted using a confocal microscope, with 15 random counts per slide. The results were reported as the Prebiotic Index value, (Bif / Total) - (Bac / Total) + (Lac / Total)-(Clos / Total), after being subjected to statistical analysis using the one-way ANOVA method.

[0141] TABLE 14.

[0142] Oligonucleotide probes used for the enumeration of bacterial groups. The Prebiotic Index of RS4 starch was significantly higher than that of the control group and the group supplemented with glucose with statistically significant. RS4 from all three types of starch, Rice flour RS4, Home flour RS4, and Hin flour RS4 stimulated the growth rate of probiotic bacteria (Lactobacillus and Bifidobacterium) more than the group of bacteria associated with gastrointestinal diseases (Bacteroides and Clostridium). The growth rates of Lactobacillus, Bifidobacterium, Bacteroides, and Clostridium were used to calculate the Prebiotic Index as illustrated in Table 15.

[0143] TABLE 15.

[0144] The prebiotic function of esterified flour as RS4. Note: * = No prebiotic function, = Prebiotic function, = Highest prebiotic function

[0145] Caco-2 Cell Line Cultivation

[0146] The human epithelial cell line originally derived from a colon carcinoma; Caco-2 was used as a model of the intestinal epithelial barrier for testing the toxicity of starch citrate. Caco-2 cells were routinely maintained in Dulbecco’s Modified Eagle Medium, DMEM with 10% fetal bovine serum and the following additions: 1% non-essential amino acids (NEAA),

[0147] 50 pM thioglycerol, 25 mg / ml gentamycin (complete medium). The cells were kept at 37°C in a humidified atmosphere containing 5% CO2. For propagation in culture flasks, Caco-2 cells were seeded in a concentration of 105cells / cm2. The medium was changed every 3 days. At 80% confluence, typically after 5 days, the cells are split 1 : 10 before further cultivation.

[0148] Trypsinize Caco-2 cells by first rinsing them with EDTA for further testing in MTT assay.

[0149] MTT Cell Viability Assay

[0150] The MTT assay was performed on Caco-2 cells. The cells were seeded in 96 well plates until the cell monolayer over 24 hours with a density of 104cells / cm2before testing with a solution of starch citrate in 2 mM potassium hydroxide in the ranges of 0.1-100 mg / L. Cells were incubated for 24, 48, and 72 hours at 37°C in a humidified atmosphere containing 5% CO2. Control groups were processed equally and incubated without starch citrate simultaneously. After treatment MTT dye (3-(4,5-dimethylthiazol-2-yl))-2,5- diphenyltetrazolium bromide, 5 mg / mL) was applied to each well for 4 hours. Cell viability was detected from the changing of tetrazolum to formazan. The absorbance was measured at 570 nm with a Microplate Reader. The experiments in each concentration treated with a solution of starch citrate were done in triplicate. Cell viability was expressed as the percentage of untreated control. All means were calculated from at least three independent experiments and are expressed as the mean ± standard deviation (SD). Analysis of statistical significance was done using the one-way analysis of variance (ANOVA). The results were considered significant if p < 0.05. By increasing the concentration of starch citrate from 1- 100 pg / mL from 24, 48, and 72 as illustrated in FIG. 15 A, 15B and 15C.

[0151] Oral glucose tolerance test (OGTT)

[0152] Four-week-old male Sprague-Dawley (SD) rats were obtained from Nomura Siam International (Bangkok, Thailand). The animals were acclimatized for seven days at the Central Animal Facility, Faculty of Science, Mahidol University (MUSC-CAF, an AAALAC-accredited facility). The health status of the animals was assessed daily by MUSC- CAF veterinarians. They were housed in a strictly hygienic conventional system with enrichment devices provided. The macroenvironment of the room was maintained at 21 ± 1°C, with 50 - 60% relative humidity, under a 12-hour light-dark cycle. Standard food (Perfect Companion Group Co., Ltd., Bangkok, Thailand) and reverse osmosis water was provided ad libitum. The protocol was approved by the Institutional Animal Care and Use Committee (IACUC), Faculty of Science, Mahidol University. All studies involving animals were conducted in accordance with relevant guidelines and regulations, including the ARRIVE guideline.

[0153] The final dosages of glucose and each RS4 were designated and prepared in corn oil (Merck Ltd., Bangkok, Thailand) as follows: 1. Glucose at 2 g / kg body weight (BW) alone.

[0154] 2. Glucose at 1.5 g / kg BW plus RS4 at 0.5 g / kg BW.

[0155] 3. Glucose at 1 g / kg BW plus RS4 at 1 g / kg BW.

[0156] 4. Glucose at 0.5 g / kg BW plus RS4 at 1.5 g / kg BW.

[0157] 5. RS4 at 2 g / kg BW alone.

[0158] Five-week-old animals were then fasted for 12 hours with access to water. Weights were measured to determine the amounts of glucose and RS4 to be administered to each animal. Initially, fasting blood samples were collected from tail tips, and glucose levels were assessed using the AlphaTrak Blood Glucose Monitoring System (Zoetis Inc., NJ, USA). Mixtures of glucose and RS4 were then administered orally using gavage needles. Glucose levels were re-assessed at five additional time points: 15, 30, 60, 90, and 120 minutes after oral gavage.

[0159] Young male SD rats that had been exposed to a regular diet for only two weeks since weaning (i.e., from 3 - 5 weeks old) were used. They were randomly divided into three major groups based on the sources of RS4 to be tested, namely RS4 from GB-Hin, GB-Hom Thomg, and rice. Within these groups, animals were further randomized into five subgroups that received mixtures of glucose and RS4 solutions at various concentrations. There were no significant differences in body weights among the animals in these five subgroups (data not shown). As expected, blood glucose levels during a 12-hour fasting period ranged between 50 and 100 mg / dL in all groups (Figure 16A - C, left panels). Upon glucose ingestion, blood glucose levels increased and reached their peaks within 30 minutes, indicating that the mechanisms for glucose absorption in the gastrointestinal (GI) tract were intact. The peaks of blood glucose levels gradually decreased as the ratios of RS4 to glucose were increased. Importantly, the peaks of blood glucose levels at 30 minutes were nearly absent in groups that received RS4 alone. Nevertheless, blood glucose levels returned to baseline in all groups within 120 minutes. The calculated AUC for all three RS4 types was significantly lower than the AUC for animals that received glucose solution alone (Figure 16A - C, right panels). These data indicate that glucose within all three types of RS4 was not available for absorption in the GI tract.

[0160] In this study, we have successfully developed starch that can withstand the digestive process in vivo. The use of these RS4 as components of daily carbohydrate sources could be beneficial for individuals who require meals with a low glycemic index. Any change made to this invention may be vividly understood and can be done by a person skilled in the field. The change may be within the scope and intent of this invention as shown in the claim attached.

Claims

1. Claims1. A method for preparing resistant starch comprising the following steps:(a) preparing monocarboxylic acid (formic acid, acetic acid, propanoic acid and quinic acid), dicarboxylic acid (succinic acid, malic acid, tartaric acid and oxalic acid), and tricarboxylic acid (citric acid and aconitic acid) aqueous solution with the concentration ranging from 10% to 40%;(b) adjusting the pH of the slurry obtained from step (a) with 5-10 M of sodium hydroxide to the pH of 2.0-3.5;(c) adding flour into the acid aqueous solution with the ratio of the flour and citric acid of 1 : 1 to 1 : 1.8, w / v to have a mixture of the flour and those weak acids to get slurry;(d) incubating the slurry obtained from the step (c) for a first period of time ranging 12-20 hours from at room temperature;(e) drying the incubated slurry obtained from the step (d) in hot air oven at 50 °C for 20-48 hours, until moisture content was less than 5%;(f) heating the dried flour obtained from the step (e), for a second period of time ranging from 3-6 hours, in hot air oven at 140-160 °C to get esterified flour;(g) washing the esterified flour obtained from the step (f) with sodium bicarbonate and distilled water to remove unreacted citric acid.(h) drying esterified flour obtained from the step (g) to reduce the moisture content of the flour to 3-7% at 50 °C for a third period ranging from 20-48 hours.

2. The method according to claim 1, wherein a concentration of citric acid, succinic acid, malic acid, and tartaric acid aqueous solution of the step (a) is preferably ranging from 10% to 25%.

3. The method according to claim 1, wherein the flour added in the step (b) is selected from one of the following flour sources comprising freeze-dried and air-dried and air-dried tapioca flour, and / or rice flour, and / or glutinous rice flour, and / or wheat flour, and / or GB- Hom Thong flour, and / or GB-Hin flour, and / or GB-Namwah flour, and / or sorghum flour, and / or mung bean flour, and / or arrowroot or a combination thereof.

4. The method according to claim 1, wherein the first period for incubating the step (d) is preferably 16 hours.

5. The method according to claim 1, wherein the drying of the slurry of the step (e) is, preferably 24 hours.

6. The method according to claim 1, wherein the drying esterified flour temperature and the second period of the step (g) is preferably at 160 °C and 5 hours.

7. The method according to claim 1, wherein the preferable washing time of the step (f) is two times washing.

8. The method according to claim 1, wherein the third period for drying the esterified flour of the step (h) is, preferably 24 hours.

9. The method according to claim 1, further comprising the step of keeping the flour citrate obtained from the step (h) in a closed container.

10. The resistant starch processed by the method of any one of claims 1-8 containing high resistant content to enzymatic digestion in a range of 3-65 %.

11. The food products prepared from the resistant starch of claim 10 having a remaining resistant starch, as RS, ranging from 7 to 34 %.

12. The method according to any one of the previous claims, wherein an alternative method for processing resistant starch is reactive extrusion and / or ribbon mixer.

13. The resistant starch according to claim 10, further containing the good texture and properties in terms of hardness, chewiness, gumminess, adhesiveness, springiness, cohesiveness, and resilience14. The resistant flour citrate product processed by the method of any one of claims 1- 5 is used as prebiotic to boost the growth of gut microbes, Lactobacillus paracasei MSMC39-1 and Bifidobacterium animalis MSMC83.

15. The resistant flour citrate product processed by the method of any one of claims 1- 5 was not toxic toward the human epithelial cell line.

16. The resistant flour citrate product processed by the method of any one of claims 1- 5, wherein Glucose within was not available for absorption in the gastrointestinal tract.

17. A method for preparing resistant starch comprising the following steps:(a) preparing and mixing 1.5 to 3.5 kilograms of flour with citric acid aqueous solution, wherein the concentration of the solution ranges from 10% to 40%, to get 10 to 20 % moisture content of the dried mixed flour;(b) reactively extruding the dried mixed flour obtained from the step (a) comprising: conveying via, mixing, and kneading within, and passing the flour through a heated barrel of extruding machine configured to have a least six heating zones to obtain extruded dried mixed flour, wherein a ratio of barrel length and barrel length diameter (L / Di) ranges from 28-36: 18-26.;(c) feeding the extruded dried mixed flour obtained from the step (b) with the feeding rate ranging from 5 to 10 kilograms per hour (kg / h) at barrel input zones, wherein the feeding is away from a metering and die zones;(d) adjusting the screw rotation speed of the extruding machine to range from 200 to 250 rpm with a torque of 40 % with the temperature from 30 to 150 °C to get starch citrate extrudate;(e) stabilizing the weight of the starch citrate extrudate, obtained from the step (d), to be constant comprising collecting, transferring in a hot air oven, and drying the starch citrate extrudate at 40 to 50 °C;(f) grounding and sieving the stabilized starch citrate extrudate obtained from the step (e) through 60- to 90-mesh sieves to get the sieved dried mixture; and(g) removing unreacted citric acid comprising: washing the sieved dried mixture obtained from the step (f) with absolute ethanol and with the 0.5-2 Molarity (M) of sodium bicarbonate to get pH 7 of resistant starch citrate product.

18. The method according to claim 17, wherein the preferred weight of flour of the step (a) ranges from 2 to 3 kilograms.

19. The method according to claim 17, wherein the concentration of citric acid aqueous solution of the step (a) is preferably ranging from 10% to 20%.

20. The method according to claim 17, wherein the preferred moisture content of the dry mixed rice flour of the step (a) is 14%.

21. The method according to claim 17, wherein the extruding machine of the step (b) is at least one of a single-screw extruder, or a twin-screw-extruder, or a co-rotating twin- screw extruder, preferably, the co-rotating twin-screw extruder.

22. The method according to claim 17, wherein a barrel length and barrel length diameter ratio (L / Di) is preferably 32: 22.

23. The method according to claim 17, wherein the feeding rate of the flour of the step (c) is preferably 8 kilograms per hour (kg / h).

24. The method according to claim 17, wherein the barrel input zone of the step (c), for the most active reaction, is preferably a barrel input zone 4.

25. The method according to claim 17, wherein the rotation speed and torque of the step (d) are preferably 250 rpm and 47%.

26. The method according to claim 17, wherein the temperature to stabilize starch citrate extrudate of the step (e) is preferably 45 °C.

27. The method according to claim 17, wherein the sieve of the step (f) is preferably an 80-mesh sieve.

28. The method according to claim 17, wherein the washing time with the absolute ethanol of the step (g) is preferably four times.

29. The method according to claim 17, wherein the washing concentration of sodium bicarbonate of the step (g) is preferably 1 M.

30. The method according to any one of claims 17-29, wherein the flour of the step (a) is selected from one of the following flour sources comprising freeze-dried and air-dried dried tapioca flour, and / or rice flour, and / or glutinous rice flour, and / or wheat flour, and / or GB-Hom Thong flour, and / or GB-Hin flour, and / or GB-Namwah flour, and / or sorghum flour, and / or mung bean flour, and / or arrowroot or a combination thereof.

31. The resistant starch citrate product processed by the method of any one of the previous claims containing high resistant content to enzymatic digestion in a range of 30-50

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