KR102441174B1 - Manufacturing method of synbiotics comprising isomalto-oligosaccharide made from rice powder - Google Patents

Abstract

The present invention relates to a method for producing a synbiotic (probiotic formulation) containing isomaltooligosaccharide using rice flour, and the prebiotic function is remarkably enhanced than before by controlling the reaction time and reaction temperature of rice flour and various enzymes. It provides a method for producing synbiotics including the isomaltooligosaccharide, dietary fiber derived from rice bran, and lactic acid bacteria by using the isomaltooligosaccharide. In addition, through this, it is possible to easily provide a pharmaceutical composition for the prevention, improvement or treatment of irritable bowel syndrome using the synbiotics, and a health functional food.

Description

Manufacturing method of synbiotics comprising isomalto-oligosaccharide made from rice powder

The present invention relates to a method for producing synbiotics as a probiotic preparation containing isomaltooligosaccharide using rice flour.

In the human intestinal tract, more than 400 kinds of bacteria live in the number of about 100 trillion per gram of the contents of the large intestine, and it is composed of a complex ecosystem that maintains a certain balance. The intestinal flora can be divided into beneficial bacteria and harmful bacteria, and the health status is controlled by the balance between them. In order to maintain health, it is necessary to maintain the intestinal flora in a state where there are many beneficial bacteria and few harmful bacteria. The flora can change depending on the state of the human body such as food, stress, hormone secretion, etc. introduced from the outside, and the enzymes and metabolites they produce are involved in the metabolism of various substances in the body, absorption of nutrients or drugs, and toxic substances affects the creation of

These beneficial bacteria in the gut are called probiotics. Prebiotics are ingredients that activate probiotics and inhibit harmful bacteria in the gut, and they play a role in creating an intestinal environment for probiotics to grow well. In other words, prebiotics serve as food for probiotics, which are microorganisms that play a beneficial role in our body. Recently, research on synbiotics, which is a mixture of these prebiotics and probiotics, has been actively researched.

The increase in beneficial intestinal flora has a direct effect on irritable bowel syndrome accompanied by diarrhea or abdominal distension among various diseases. Irritable Bowel Syndrome is a generic term for symptoms caused by dysfunction due to overactive contraction of the colon muscle without other diseases or anatomical abnormalities. Various studies have reported that this is mainly caused by stress.

Rice has been functioning as a 'source of energy' and 'a foundation of culture' since the time humans used stone tools. Although rice is the staple food, rice cultivation is known to have labor-intensive characteristics and nutritional integrity. About 20% of the world's arable land is paddy field, and rice has a higher population buoyancy compared to other crops, so the area where rice is the main staple has a high population density. Due to its low population support capacity, only 10% of the world's population uses it as a stock.

Rice is the main energy source for Koreans, and 30-40% of the daily energy required by adults is from rice. Rice contains about 10 nutritional components such as carbohydrates, proteins, fats, dietary fiber, and minerals, which are mainly contained in rice bran and rice bran. This is actively progressing, and the development of varieties and processing technologies that increase the content of functional ingredients in rice through traditional breeding, biotechnology, and germination treatment are accelerating.

However, due to the diversification of dietary habits and changes in eating habits, rice consumption is on a downward trend by 1.6~1.2kg per year. In 2014, consumption per person decreased to 65.1kg.

In addition, the surplus of domestic rice is increasing due to rice coming in through MMA (mandatory import quantity). Although this has decreased slightly, the rice inventory has continued to increase since 2012, and the rice inventory in September 2015 increased by 95% compared to 2012 to 1.36 million tons. From 313.1 billion (2012) to 429.7 billion (September 2015), the economic loss continues to increase. However, rice used for manufacturing processed foods only accounts for about 6% of domestic production, so there is a strong need for the rice processing industry for purposes other than rice as food. Therefore, it is necessary to form a new consumption pattern such as increased consumption of processed rice products along with convenience food, well-being, LOHAS, and the environment, which are recent food consumption patterns.

Dietary fiber refers to an indigestible high molecular fiber component that is not broken down by digestive enzymes in the body, and is mainly distributed in the cell wall of plant cells or the shell of plant seeds. Since dietary fiber is not absorbed by the body, it was recognized as of no nutritional value. However, recently, interest in functional foods has increased, and physiological functions different from the six major nutrients of carbohydrates, proteins, fats, vitamins, minerals, and water have been recognized, so it is called the 7th nutrient. Physiological functions of dietary fiber include smooth bowel movements, aid in weight loss, inhibition of fat absorption, inhibition of fat synthesis, and decomposition of body fat. Dietary fiber is not digested in the body and reaches the intestine where it becomes a nutrient for lactic acid bacteria. In addition, it promotes the movement of the large intestine, shortens the time for feces to pass through the large intestine, and increases the amount of bowel movement to facilitate bowel movements. Recently, along with the proliferation of rice processing plants, the production of rice bran, a by-product of rice, is also increasing. Although rice bran contains a large amount of human active ingredients such as protein and dietary fiber compared to white rice, it is easily deteriorated and thus used as feed or discarded. Therefore, the technology of extracting dietary fiber from rice bran and using it as a food additive is being developed more and more, and it is necessary to develop a product using the dietary fiber derived from rice bran.

Isomaltooligosaccharides are branched oligosaccharides in which glucose molecules have α-1,6 bonds, and are produced by the transfer reaction of maltose or maltooligosaccharides by a sugar transferase. Maltooligosaccharide is a straight-chain oligosaccharide in which 3 to 10 glucose units are linked by α-(1→4) bonds. Representative examples of isomaltooligosaccharides include isomaltose, panose, isomaltotriose, and isomaltotetraose. In addition, isomaltooligosaccharide can be widely applied to food due to its thermal stability, low viscosity, and high moisture-retaining properties, and it is not fermented by yeast, so it can be used as a sweetener for baking, alcohol, and seasoning. The physiological functions of isomaltooligosaccharide include lowering the glycemic index because it is slowly decomposed by intestinal enzymes, and has a prebiotic function that promotes intestinal function by proliferating beneficial intestinal bacteria, improves immune function, and improves the bioavailability of some minerals. It has been reported to have effects such as increasing blood pressure, promoting the metabolism of triglycerides, and improving blood cholesterol.

On the other hand, the present inventors were researching a composition for improving rice consumption and prebiotics, synbiotics as a probiotic preparation using the prebiotics, and irritable bowel syndrome, reaction time and reaction temperature of rice flour and various enzymes, etc. It is possible to manufacture isomaltooligosaccharide with significantly enhanced prebiotic function than before by controlling , has completed the present invention by preparing a composition having an improvement and therapeutic efficacy.

Republic of Korea Patent Registration No. 10-1628332 (Title of the invention: Rice sugar manufacturing method and rice sugar thereof, Applicant: Kim Dong-yoon, Registration date: June 01, 2016) Republic of Korea Patent Registration No. 10-0387286 (Title of the invention: manufacturing method of isomaltooligosaccharide, Applicant: Samyang Genex Co., Ltd., Registration date: May 30, 2003) Republic of Korea Patent Registration No. 10-2012440 (Title of the invention: manufacturing method of isomaltooligosaccharide composition, Applicant: Ingredion Korea Co., Ltd., Registration date: August 13, 2019) Republic of Korea Patent No. 10-1856751 (Title of the invention: Manufacturing method of mixed sugar composition containing isomaltooligosaccharide, Applicant: Samyang Corporation, Registration date: May 03, 2018) Republic of Korea Patent Registration No. 10-1776125 (Title of the invention: Rice saccharified beverage using complex enzyme and manufacturing method thereof, Applicant: Lotte Chilsung Beverage Co., Ltd., Registration date: September 01, 2017) Republic of Korea Patent No. 10-1547944 (Title of the invention: Confectionery for improving obesity and manufacturing method thereof, Applicant: Seunghee Lee, Registration date: August 21, 2015) Republic of Korea Patent Publication No. 10-1992-0011366 (Title of the invention: multi-functional fermented milk composition, Applicant: Cheiljedang Co., Ltd., publication date: July 24, 1992) Korean Patent Laid-Open Patent No. 10-2019-0128087 (Title of Invention: Multi-fiber prebiotic preparation for digestive health, weight control, immune enhancement and health improvement, Applicant: Kibow Biotech Inc., Publication date: 2019 November 14th)

An object of the present invention is to provide a method for producing a probiotic preparation containing isomaltooligosaccharide using rice flour.

The present invention relates to a method for producing synbiotics containing isomaltooligosaccharide using rice flour. Synbiotics are probiotics containing prebiotics.

The method is preferably as follows.

That is, (first step) to prepare a rice flour slurry by adding CaCO ₃ so that calcium ions are 60 to 80 ppm to the rice flour dispersion, and adjusting the pH to 6.0 to 6.4 while adding 0.8 to 1.2 N of HCl dropwise;

(Step 2) The temperature of the rice flour slurry is raised and α-amylase is added at 58 to 62 ° C. After the enzyme is added, the temperature of the rice flour slurry is continuously raised to maintain the temperature at 90 to 95 ° C. liquefying the slurry;

(Third step) β-amylase, pullulanase and transglucosidase were added to the rice liquefied solution while maintaining the temperature by lowering the temperature of the rice flour liquefied solution obtained in the second step to 55-60° C. adding and reacting to saccharify the liquid to obtain isomaltooligosaccharide;

(Step 4) filtering and concentrating the isomaltooligosaccharide obtained in step 3 to obtain isomaltooligosaccharide having a sugar content of 70 to 80 Brix;

(Step 5) pulverizing the concentrated isomaltooligosaccharide;

(Sixth step) mixing the isomaltooligosaccharide obtained in step 5, dietary fiber derived from rice bran and lactic acid bacteria powder; may include.

In the first step, the rice flour dispersion is characterized in that 300 to 500 parts by weight of water based on 100 parts by weight of rice are mixed and dispersed.

In the first step, the particle size of the rice flour is 180 μm or less, and the moisture content is preferably 14% by weight or less. Preferably, the particle size is 10 to 180 μm, and the moisture content may be 12 to 14 wt %. When the particle size of rice flour exceeds 180 μm, saccharification reaction does not occur well, which is undesirable. may not happen

The α-amylase used in the second step may have heat resistance with optimal enzymatic activity at a temperature of 90 to 100°C. For the production of this enzyme, any strain optimized for enzyme production may be used, but in the present invention, it is preferable to use one produced from a Bacillus licheniformis strain.

In the second step, the α-amylase enzyme is added at 58 to 62° C. to prevent rapid gelatinization of rice flour. When α-amylase enzyme is added after raising the temperature to 90~95℃, which is the optimum reaction temperature, gelatinization occurs rapidly before the enzymatic reaction, and rice flour is agglomerated to form a mass in the dispersion. In this case, the enzyme reaction inside the mass occurs It's not good because it can't happen.

It is appropriate that the enzymatic reaction time at 90 to 95° C. of the second step is for 1 to 3 hours.

The α-amylase enzyme of the second step may contain 0.05 to 0.1 parts by weight based on 100 parts by weight of rice flour.

The enzymatic reaction of the third step is preferably carried out at 55 ~ 58 ℃.

The enzyme used in the third step, β-amylase, has optimal activity at 55 to 65 ° C, pullulanase at 55 to 60 ° C, and transglucosidase at 58 to 62 ° C. For the preparation, any strain optimized for enzyme production may be used, but preferably, β-amylase produced from a Bacillus subtilis amyloliquefaciens strain, pullulus produced from a Bacillus acidopuluriticus strain The use of transglucosidase produced from Lanase and Aspergillus niger strains is most suitable for glycosylation.

The reaction time of the enzyme in the third step is preferably 30 to 40 hours.

The content of each enzyme in the third step may be 0.001 to 0.002 parts by weight of β-amylase, 0.05 to 0.1 parts by weight of pullulanase, and 0.07 to 0.2 parts by weight of transglucosidase based on 100 parts by weight of rice flour. When the enzyme is treated at this concentration, the content of glucose is reduced and the content of isomaltooligosaccharide having a high degree of polymerization is increased. At this time, if the content of each enzyme is less than the range, saccharification does not occur well, so the production of isomaltooligosaccharide is not good. not.

The composition containing isomaltooligosaccharide obtained in the third step is characterized in that it is 16-18 Brix, isomaltose 30-40 g/L, panose 8-15 g/L, isomal 15-25 g/L of isomaltotriose, 5-10 g/L of isomaltotetraose, 0.5-2 g/L of isomaltopentaose, and 60-95 g/L of total isomaltooligosaccharides When the total saccharides excluding L, water and other minor components are 100% by weight, the content of isomaltooligosaccharide may be 50 to 60% by weight.

The isomaltooligosaccharide obtained after concentration in the fourth step is isomaltose (isomaltose) 125 ~ 166 g / L, panose (panose) 33 ~ 63 g / L, isomaltotriose (isomaltotriose) 63 ~ 105 g / L, Isomaltotetraose 21-42 g/L, isomaltopentaose 1-8 g/L, total isomaltooligosaccharides 250-395 g/L, total sugars excluding water and other minor ingredients When referring to 100% by weight, the content of isomaltooligosaccharide may be 50 to 60% by weight.

The isomaltooligosaccharide obtained after concentration in the fourth step may be 70 to 80 Brix.

In the sixth step, based on 1 g of isomaltooligosaccharide powder, 0.5 to 2 g of rice bran-derived dietary fiber and 0.02 to 0.04 g of lactic acid bacteria powder may be included, and in this case, the total number of lactic acid bacteria is preferably 1×10 ⁸ to 1×10 ¹¹ CFU, preferably about 3×10 ⁸ CFU.

The lactic acid bacteria of the sixth step are Lactobacillus rhamnosus ( Lactobacillus rhamnosus ), Lactobacillus acidophilus ( Lactobacillus acidophilus ), Lactobacillus delbruecky subsp. Bulgaricus ( Lactobacillus delbrueckii subsp . bulgaricus ) may be a mixed lactic acid bacteria, and these may be mixed in a weight ratio of 1:0.5:0.5 to 1:2:2. Each lactic acid bacterium can be used in a powdered form of 1×10 ⁸ to 1×10 ¹¹ CFU/g.

As for the dietary fiber derived from rice bran in the sixth step, it is preferable to use 20-30% by weight of insoluble dietary fiber and 2-10% by weight of soluble dietary fiber. The rice bran-derived dietary fiber contains water and cellulase in rice bran may be obtained by adding 100 to 200 parts by weight of water based on 100 parts by weight of rice bran so that it is 30 to 50° C., then treated with 0.005 to 0.1 parts by weight of cellulase and reacted for 1 to 3 hours. can Preferably, the dietary fiber derived from rice bran may be prepared by adding 100 parts by weight of rice bran and 100 parts by weight of water to 37° C., treating 0.01 parts by weight of cellulase and reacting for 2 hours.

The present invention may provide a probiotic preparation prepared by the above method.

It is also available as a pharmaceutical composition or health functional food for preventing, treating, and improving irritable bowel syndrome containing the probiotic preparation.

In the present invention, irritable bowel syndrome may be a diarrhea-predominant disease.

In the pharmaceutical composition for the prevention or treatment of irritable bowel syndrome, it is best to use it as a composition for oral administration itself in consideration of the fact that it is a probiotic preparation, and various excipients may be added as necessary. The dosage of the pharmaceutical composition of the present invention will vary depending on the age, sex, and weight of the subject to be treated, the specific disease or pathological condition to be treated, the severity of the disease or pathological condition, the route of administration, and the judgment of the prescriber. Dosage determination based on these factors is within the level of one of ordinary skill in the art, and dosages generally range from 0.01 mg/kg/day to approximately 2000 mg/kg/day. A more preferred dosage is 1 mg/kg/day to 500 mg/kg/day. Administration may be administered once a day, or may be administered in several divided doses. The above dosage does not limit the scope of the present invention in any way. The pharmaceutical composition of the present invention may be administered to mammals such as mice, livestock, and humans by various routes.

As a health functional food for the prevention or improvement of irritable bowel syndrome, the probiotic preparation can be provided as it is, and it can also be provided as various food toppings and garnishes. For example, it can be used as an additive for food, such as meat, sausage, bread, candies, snacks, ice cream, dairy products, gum, and vitamin complexes.

The present invention relates to a method for producing synbiotics (probiotics preparation) containing isomaltooligosaccharide using rice flour, and to isomaltooligosaccharide prepared using starch by controlling the reaction time and reaction temperature of rice flour and various enzymes. In comparison, it is possible to manufacture isomaltooligosaccharides, which are nutritionally excellent as all ingredients of rice are saccharified, but with significantly enhanced prebiotic function than before. In addition, the rice flour used in the present invention has a higher raw material price compared to corn used as the main raw material for starch, but compared to separately producing corn starch, the production cost is lower and the production cost is simpler because it only needs to be powdered. As it is possible to produce rice flour, it is more advantageous in terms of cost/time in manufacturing isomaltooligosaccharide using starch prepared from corn. In addition, while corn, which is used as the main raw material for starch, contains many GMO products, rice has the advantage of providing safe food because it does not use GMO crops.

In addition, the present invention provides a method for producing synbiotics comprising the isomaltooligosaccharide, rice bran-derived dietary fiber and lactic acid bacteria, and through this, a pharmaceutical composition for preventing, improving or treating irritable enteritis syndrome using the synbiotics, Health functional food can be easily provided.

1 is a photograph of a synbiotic, that is, a probiotic formulation of Preparation Example 1 of the present invention.

Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, it is provided so that this disclosure will be thorough and complete, and will fully convey the spirit of the invention to those skilled in the art.

<Example 1. Isomaltooligosaccharide prepared through step-by-step enzyme treatment>

For the liquefaction reaction of rice flour, 400 g of distilled water per 100 g of rice flour was mixed and dispersed by mixing rice flour (white rice) and distilled water in a weight ratio of 1:4. For stabilization of the glycosylation enzyme, 0.035 g of CaCO ₃ was added so that the calcium ion was 70 ppm, and the pH was adjusted to 6.2 using 1N HCl.

α-Amylase produced from the Bacillus licheniformis strain to prevent rapid gelatinization of the rice flour itself when the temperature of the rice dispersion reaches 60°C while shaking the thus-made rice flour dispersion at room temperature (25°C) in a water bath at 60°C. (Termamyl 2X) was added. Next, the temperature was raised to 95° C. and allowed to react for 2 hours to prepare a sample in a dispersion state in a liquefied state.

At this time, α-Amylase produced from the Bacillus licheniformis strain was used in an amount of 0.075 g compared to 100 g of rice flour.

After liquefaction, the liquefied solution was cooled to 55° C. for optimal conditions for the saccharification enzyme to act, and the pH was also adjusted to 5.0 with 1 N HCl or 1 N NaOH.

For saccharification, β-Amylase (Maltogenase L) produced from Bacillus subtilis amyloliquefaciens strain, Pullulanase (Promozyme D2) produced from Bacillus acido pulluliticus strain, Transglucosidase produced from Aspergillus niger strain (Transglucosidase L) was added to the rice liquefied solution, and as an optimal enzyme concentration, 0.0015 g of β-Amylase (Maltogenase L), 0.07 g of Pullulanase (Promozyme), and 0.1 g of Transglucosidase (Transglucosidase L) compared to 100 g of rice flour were added. When the enzyme is treated at this concentration, the content of glucose is reduced and the content of isomaltooligosaccharide having a high degree of polymerization is increased.

In this way, three kinds of enzymes were added and reacted for 36 hours while maintaining 55° C., to obtain a composition containing 75.36 g/L of total isomaltooligosaccharides. The isomaltooligosaccharide prepared under optimal conditions was 18 brix, and isomaltose 35.11 g/L, panose 11.79 g/L, isomaltotriose 19.95 g/L, isomaltotetraose 7.46 g/L, and isomaltopentaose 1.05 g/L were produced. The content of the oligosaccharide is found to be 56.37 wt%.

Enzymes for each liquefaction and saccharification used in this process are shown in Table 1 below.

Enzyme Optimal conditions Company Manufacture Origin Temp. (℃) pH Termamyl2X
(α-Amylase) 90-100 6.0~6.4 Novozymes Bacillus licheniformis
( Bacillus licheniformis ) Maltogenase L
(β-Amylase) 55~65 4.5~5.5 Novozymes bacillus
subtilis amyloliquefaciens
( Bacillus subtilisamyloliquefaciens ) PromozymeD2
(Pullulanase) 55~60 4.2~5.4 Novozymes Bacillus acidopuluriticus
( Bacillus acidopullulyticus ) Transglucosidase L
(Transglucosidase) 58-62 4.8~5.2 Amano Aspergillus niger
( Aspergillus niger )

The saccharified rice solution that had undergone saccharification and transfer for 36 hours with β-Amylase (maltogenase L), Pullulanase (promozyme D2), and Transglucosidase L enzymes was filtered with filter paper, and the filtered sugar solution was concentrated with a vacuum concentrator to a sugar content of 75 brix. prepared.

<Example 2. Isomaltooligosaccharide prepared by controlling the enzyme reaction time>

Example 2-1

A saccharide composition was prepared in the same manner as in Example 1, except that the enzymatic reaction time at 95°C was 1 hour.

Example 2-2

A saccharide composition was prepared in the same manner as in Example 1, except that the enzymatic reaction time at 95°C was 3 hours.

Example 2-3

A saccharide composition was prepared in the same manner as in Example 1, except that the reaction time for the three enzymes was 30 hours.

Example 2-4

A saccharide composition was prepared in the same manner as in Example 1, except that the reaction time for the three enzymes was 40 hours.

<Comparative Example 1. Sugar composition prepared by adjusting the type of each enzyme>

Comparative Example 1-1

A saccharide composition such as isomaltooligosaccharide was prepared in the same manner as in Example 1, except that the first enzyme treatment step was omitted and three enzyme treatment steps were immediately performed to saccharify the rice flour dispersion to prepare a sugar composition.

Comparative Example 1-2

A saccharide composition was prepared in the same manner as in Example 1, except for β-Amylase (Maltogenase L) produced from the Bacillus subtilis amyloliquefaciens strain instead of the second three kinds of enzyme treatment. Same as the three enzymes of Example 1. It was treated with rice liquefied solution by weight.

Comparative Example 1-3

A saccharide composition was prepared in the same manner as in Example 1, but instead of the second three kinds of enzyme treatment, only Pullulanase (Promozyme D2) produced from the Bacillus acidopuluriticus strain was liquefied rice with the same weight as the three enzymes of Example 1. treated with the liquid.

Comparative Example 1-4

A saccharide composition was prepared in the same manner as in Example 1, but only Transglucosidase L produced from the Aspergillus niger strain was treated with the same weight as the three enzymes of Example 1 in the rice liquefied solution instead of the second three kinds of enzyme treatment.

Comparative Example 1-5

A saccharide composition was prepared in the same manner as in Example 1, but instead of the second three kinds of enzyme treatment, β-Amylase (Maltogenase L) produced from the Bacillus subtilis amyloliquefaciens strain, The produced Pullulanase (Promozyme D2) two enzymes were mixed at a weight ratio of 1:1 and treated with the same weight as the three enzymes of Example 1 in the liquefied rice.

Comparative Example 1-6

A saccharide composition was prepared in the same manner as in Example 1, but instead of the second three kinds of enzyme treatment, β-Amylase (Maltogenase L) produced from Bacillus subtilis amyloliquefaciens strain, Transglucosidase produced from Aspergillus niger strain L 2 enzymes were mixed in a weight ratio of 1:1 and treated with the same weight as the 3 enzymes of Example 1 in the liquefied rice.

Comparative Example 1-7

A saccharide composition was prepared in the same manner as in Example 1, except that, instead of treating the second three kinds of enzymes, Pullulanase (Promozyme D2) produced from the Bacillus acidopuluriticus strain, and Transglucosidase L two enzymes produced from the Aspergillus niger strain was mixed in a weight ratio of 1:1 and treated with the rice liquefied solution at the same weight as the three enzymes of Example 1.

<Comparative Example 1. Isomaltooligosaccharide prepared by controlling the reaction time of each enzyme treatment>

Comparative Example 2-1

Isomaltooligosaccharide was prepared in the same manner as in Example 1, but the enzymatic reaction time at 95°C was 4 hours.

Comparative Example 2-2

Isomaltooligosaccharide was prepared in the same manner as in Example 1, but the enzyme reaction time at 95°C was 0.5 hours.

Comparative Example 2-3

Isomaltooligosaccharide was prepared in the same manner as in Example 1, but the reaction time of the three enzymes was 20 hours.

Comparative Example 2-4

Isomaltooligosaccharide was prepared in the same manner as in Example 1, but the reaction time of the three enzymes was set to 50 hours.

Comparative Example 2-5

Prepare isomaltooligosaccharide in the same manner as in Example 1, except that in the first enzyme treatment step, the temperature was raised up to 55 ° C and α-amylase derived from Aspergillus niger (Fungamyl 800 L, 5.0-6.0, 53~58°C) and maintained at 55°C for 2 hours, followed by a second saccharification enzyme treatment step.

※ If only the first enzymatic treatment step is performed and the three enzymatic treatment steps are omitted, saccharification reaction does not occur and the experiment is not performed. In addition, in the process of adding α-Amylase produced from the Bacillus licheniformis strain in the first enzyme treatment step immediately after raising the temperature to 95° C. without treatment at 60° C. Not tested.

<Experimental Example 1. Evaluation of the effect in the defecation model (Restraint stress-induced fecal pellet output model) by restraint stress>

Example 1 and Comparative Example 1 of isomaltooligosaccharide or saccharides of irritable bowel syndrome improvement function by restraint stress defecation model (Restraint stress-induced fecal pellet output model) [S. Kobayashi et al., Jpn. J. Pharmcaol., 86, p 281-288, 2001].

As experimental animals, male Sprague-Dawley rats (Charles River) weighing 250-300 g were used, and the SD rats were subjected to a temperature of 25° C., a humidity of 50%, and a day-night cycle of 12:12 hours in an animal room controlled for 12 hours per cage. Two were bred. Water and feed were freely accessible, and after 5 days of acclimatization, a restraint experiment was performed.

On the day of the experiment, restraint-induced fecal pellet output was measured in SD rats using a restraint cage. To this end, each composition of each of Examples 1 and 2 was dissolved in 0.5% (w.v) CMC aqueous solution and orally administered at a concentration of 300 mg/kg, and the experimental animals were placed in a calibration frame. At this time, care was taken not to stress the animals due to administration. Immobility within the cage puts the animal under bondage stress and initiates defecation.

The pattern and number of stools were measured at 60-minute intervals for 4 hours, and the results are shown in Table 2. For statistical treatment, significance was tested at the level of p<0.01 (**) or p<0.001 (***) using Student's t-test.

experimental group number of bowel movements number of diarrhea stomach condition Bondage stress untreated group 2 0 normal bondage stress treatment group 10 9 bloating Bondage stress treatment group + isomaltooligosaccharide of Example 1 2 0 normal Bondage stress treatment group + isomaltooligosaccharide of Example 2-1 3 0 normal Bondage stress treatment group + isomaltooligosaccharide of Example 2-2 3 0 normal Bondage stress treatment group + isomaltooligosaccharide of Example 2-3 3 0 normal Bondage stress treatment group + isomaltooligosaccharide of Example 2-4 3 0 normal Bondage stress treatment group + saccharides of Comparative Example 1-1 8 8 bloating Bondage stress treatment group + saccharides of Comparative Example 1-2 4 4 bloating Bondage stress treatment group + saccharides of Comparative Examples 1-3 5 3 bloating Bondage stress treatment group + saccharides of Comparative Examples 1-4 5 3 bloating Bondage stress treatment group + saccharides of Comparative Examples 1-5 3 3 bloating Bondage stress treatment group + saccharides of Comparative Examples 1-6 3 3 bloating Bondage stress treatment group + saccharides of Comparative Examples 1-7 4 3 bloating Bondage stress treatment group + isomaltooligosaccharide of Comparative Example 2-1 7 4 bloating Bondage stress treatment group + isomaltooligosaccharide of Comparative Example 2-2 3 3 bloating Bondage stress treatment group + isomaltooligosaccharide of Comparative Example 2-3 6 3 bloating Bondage stress treatment group + isomaltooligosaccharide of Comparative Example 2-4 3 3 bloating Bondage stress treatment group + isomaltooligosaccharide of Comparative Example 2-5 6 4 bloating Bondage stress treatment group + commercially available Jocheong 8 7 bloating Bondage stress treatment group + commercially available oligosaccharides 8 6 bloating Bondage stress treatment group + commercially available isomaltooligosaccharide 5 4 bloating

As a result, as shown in Table 2 above, in the group treated with isomaltooligosaccharide of the present invention, the number of bowel movements (fecal pellet output) caused by constraint stress was significantly reduced compared to the isomaltooligosaccharide untreated group (water), and the constraint stress was reduced. It was confirmed that feces were excreted similarly to the general group, which was not applied at all. The state of stool and distension of the abdomen were also compared, the group administered with isomaltooligosaccharide of the present invention was similar to the general group, but the group administered with the saccharide of Comparative Example 1 was mostly checked for diarrhea, and the state of abdominal distension was also It was serious. In particular, from these results, it can be seen that crude oil, oligosaccharides, and commercially available isomaltooligosaccharides alone have little effect on reducing abdominal distension, diarrhea, or frequent bowel movements.

<Experimental Example 2. Confirmation of the culture efficacy of intestinal bacteria>

In Experimental Example 1, feces obtained from SD rats for 4 hours were collected for each treatment group, and 1 g of the feces was diluted to a total of 10 ml by adding physiological saline, and used as a raw material sample for the experiment. The liquid obtained by diluting each sample again by a 10-fold dilution method is incubated in MRS agar medium, which is a medium for lactobacillus culture, at 37°C for 20 hours, and the number of colonies generated is counted. did. The total number of lactic acid bacteria in the feces was described in comparison with Table 3 below, based on a value of 100 for the untreated group under restraint stress.

experimental group Lactobacillus (fold) in 1g/10ml dilution of feces Bondage stress untreated group 100 bondage stress treatment group 0.1 Bondage stress treatment group + isomaltooligosaccharide of Example 1 116.2 Bondage stress treatment group + mixture of Example 2-1 84.1 Bondage stress treatment group + mixture of Example 2-2 82.3 Bondage stress treatment group + saccharides of Comparative Example 1-1 0.4 Bondage stress treatment group + saccharides of Comparative Example 1-2 2.2 Bondage stress treatment group + saccharides of Comparative Examples 1-3 1.5 Bondage stress treatment group + saccharides of Comparative Examples 1-4 2.5 Bondage stress treatment group + saccharides of Comparative Examples 1-5 9.4 Bondage stress treatment group + saccharides of Comparative Examples 1-6 8.3 Bondage stress treatment group + saccharides of Comparative Examples 1-7 9.2 Bondage stress treatment group + isomaltooligosaccharide of Comparative Example 2-1 1.2 Bondage stress treatment group + isomaltooligosaccharide of Comparative Example 2-2 15.3 Bondage stress treatment group + isomaltooligosaccharide of Comparative Example 2-3 1.2 Bondage stress treatment group + isomaltooligosaccharide of Comparative Example 2-4 12.6 Bondage stress treatment group + isomaltooligosaccharide of Comparative Example 2-5 5.6 Bondage stress treatment group + commercially available Jocheong 5.1 Bondage stress treatment group + commercially available oligosaccharides 5.4 Bondage stress treatment group + commercially available isomaltooligosaccharide 4.3

The results of Table 3 support that the isomaltooligosaccharide of the present invention improves the useful microflora by enhancing or restoring the number of intestinal lactic acid bacteria for defecation abnormalities according to restraint stress. Therefore, through these results, it can be confirmed that the isomaltooligosaccharide prepared in the present invention is a composition suitable for use as a prebiotic for regulating the proliferation of intestinal probiotics. In addition, it can be seen from the above results that the control of the total number of lactic acid bacteria is not well with only commercially available isomaltooligosaccharides, oligosaccharides, and crude oil.

<Preparation Example 1. Preparation of Synbiotics (Probiotics Formulation)>

In order to formulate probiotics, the isomaltooligosaccharide of Example 1, which had the best function as prebiotics, was powdered, and rice bran-derived dietary fiber powder and three types of lactic acid bacteria were mixed to prepare a probiotic formulation as follows. and sealed (isomaltooligosaccharide powder: dietary fiber = 1:1).

Information on the dietary fiber powder derived from rice bran is described in Table 4 below, and the manufacturing information of the probiotic formulation is described in Table 5.

Item unit result moisture content % 8.4 ash % 1.5 george % 3.0 crude protein % 2.1 Dietary Fiber % 30 crude carbohydrate % 55

Raw material Where to buy 1 sachets content Characteristics of raw materials Rice bran-derived dietary fiber powder Haanxi Undersun
Biomedthech Co. 4 g Contains 25.1w% of insoluble dietary fiber and 4.9w% of soluble dietary fiber Example 1
Isomaltooligosaccharide powder self-production 4 g - Lactobacillus Lactobacillus rhamnosus CTC Bio 0.04 g Total number of lactic acid bacteria
5.13 × 10 ¹⁵ CFU/g Lactobacillus acidophilus Vision Biocam 0.04 g Total number of lactic acid bacteria
1.82 × 10 ¹⁶ CFU/g Lactobacillus delbrueckii subsp . bulgaricus CTC Bio 0.04 g Total number of lactic acid bacteria
6.45 × 10 ¹³ CFU/g

As shown in Table 4, the components of 8.12 g of synbiotics prepared by mixing each raw material were analyzed again and shown in Table 6 below.

The rice bran-derived dietary fiber was prepared by adding 100 g of water based on 100 g of rice bran to 37° C., then treating 0.01 g of cellulase and reacting for 2 hours.

Furtherance Composition evaluation of Synbiotics products number of lactic acid bacteria 3 × 10 ⁸ CFU/g Dietary Fiber Soluble dietary fiber (%) 2.4 Insoluble dietary fiber (%) 13.2 total dietary fiber
content (%) 15.6 oligosaccharide glucose 0.07 pannose 5.76 maltose 9.72 maltotetraose 7.40 isomaltose 1.0 maltopentaose 6.79 maltotriose 12.87 maltohexaose 4.50 Total content (%) 48.11

[Manufacture of synbiotics with different content of raw materials]

<Production Example 2>

Synbiotics were prepared in the same manner as in Preparation Example 1 except that 5 g of isomaltooligosaccharide powder of Example 1 and 3g of dietary fiber derived from rice bran were used (isomaltooligosaccharide powder: dietary fiber = 1:0.6).

<Production Example 3>

A probiotic formulation was prepared in the same manner as in Preparation Example 1, except that 3 g of isomaltooligosaccharide powder of Example 1 and 4g of dietary fiber derived from rice bran were used (isomaltooligosaccharide powder: dietary fiber = 1:1.6).

<Comparative Preparation Example 1>

A probiotic formulation was prepared in the same manner as in Preparation Example 1 except that 6 g of isomaltooligosaccharide powder of Example 1 and 2g of dietary fiber derived from rice bran were used (isomaltooligosaccharide powder: dietary fiber = 1:0.3).

<Comparative Preparation Example 2>

A probiotic formulation was prepared in the same manner as in Preparation Example 1, except that 2 g of isomaltooligosaccharide powder of Example 1 and 6g of dietary fiber derived from rice bran were used (isomaltooligosaccharide powder: dietary fiber = 1:3).

<Comparative Preparation Example 3>

Probiotics were prepared in the same manner as in Preparation Example 1, but sugar was used instead of the isomaltooligosaccharide powder of Example 1.

<Comparative Preparation Example 4>

Probiotics were prepared in the same manner as in Preparation Example 1, but commercially available soy sauce was used instead of the isomaltooligosaccharide powder of Example 1.

<Comparative Preparation Example 5>

Probiotics were prepared in the same manner as in Preparation Example 1, but a commercially available oligosaccharide was used instead of the isomaltooligosaccharide powder of Example 1.

<Comparative Preparation Example 6>

Probiotics were prepared in the same manner as in Preparation Example 1, but a commercially available oligosaccharide was used instead of the isomaltooligosaccharide powder of Example 1.

<Comparative Preparation Example 6>

Probiotics were prepared in the same manner as in Preparation Example 1, but rice bran-derived dietary fiber was further added instead of the isomaltooligosaccharide of Example 1 (a total of 8 g of dietary fiber).

<Experimental Example 3. Confirmation result of lactic acid bacteria survival rate in gastrointestinal conditions>

Using hydrochloric acid, the pH of 100 ml of Lactobacilli MRS broth (BD) was adjusted to 3.0 and then sterilized, and 0.4 g of pepsin (Sigma P7000, 250 Units/mg) was mixed with 100 ml of sterilized MRS broth to prepare an artificial gastric juice medium. . In 100 ml of the prepared artificial gastric juice medium, 0.5 g of each of the probiotic formulation powders of Preparation Examples 1 to 3 and Comparative Preparation Examples 1 to 6 were mixed. Each mixture was immediately taken, diluted with physiological saline, and 1 ml of the diluted solution was dispensed in a Petridish, and 20 ml of sterilized Bromo cresol purple agar (BD) medium was added to harden. Colonies of Petridish cultured for 48 hours in a stationary incubator at 37° C. were counted and compared with the initial number of bacteria, and are shown in Table 7 below.

treatment group Bacterial survival rate (%) Preparation Example 1 52.8 Preparation 2 56.3 Preparation 3 54.2 Comparative Preparation Example 1 15.4 Comparative Preparation Example 2 15.2 Comparative Preparation Example 3 3.61 Comparative Preparation Example 4 9.91 Comparative Preparation Example 5 0.00 Comparative Preparation Example 6 9.24

As a result, the survival rate of the probiotic preparation of the present invention in the state of artificial gastric juice with hydrochloric acid was 50% or more, but it was confirmed that the survival rate of the probiotics prepared under other conditions was very low in the state of gastric juice.

Claims (8)

(Step 1) preparing a rice flour slurry by adding CaCO ₃ to the rice flour dispersion so that calcium ions are 60 to 80 ppm, and adjusting the pH to 6.0 to 6.4 while adding 0.8 to 1.2 N of HCl dropwise;
(Second step) The temperature of the rice flour slurry was raised and α-amylase was added at 58 to 62 ° C. After the enzyme was added, the temperature of the rice flour slurry was continuously raised to 90 to 95 ° C. The temperature was maintained for 1 to 3 hours. liquefying the rice flour slurry by enzymatic reaction in the;
(Third step) β-amylase, pullulanase and transglucosidase were added to the rice liquefied solution while maintaining the temperature by lowering the temperature of the rice flour liquefied solution obtained in the second step to 55-60° C. obtaining an isomaltooligosaccharide by adding and reacting for 30 to 40 hours to saccharify the liquid;
(Step 4) filtering and concentrating the isomaltooligosaccharide obtained in step 3 to obtain isomaltooligosaccharide having a sugar content of 70 to 80 Brix;
(Step 5) pulverizing the concentrated isomaltooligosaccharide;
(Step 6) mixing isomaltooligosaccharide obtained in step 5, dietary fiber derived from rice bran and lactic acid bacteria powder;
As a method for producing a probiotic formulation containing isomaltooligosaccharide using rice flour, comprising:
The rice flour dispersion is dispersed by mixing 300 to 500 parts by weight of water based on 100 parts by weight of rice flour,
The α-amylase enzyme of the second step contains 0.05 to 0.1 parts by weight based on 100 parts by weight of rice flour,
The content of each enzyme in the third step is based on 100 parts by weight of rice flour, β-amylase 0.001 to 0.002 parts by weight, pullulanase 0.05 to 0.1 parts by weight, and transglucosidase 0.07 to 0.2 parts by weight.
In step 6, based on 1 g of isomaltooligosaccharide, 0.5 to 2 g of rice bran-derived dietary fiber and 0.02 to 0.04 g of lactic acid bacteria powder containing 1 × 10 ⁸ to 1 × 10 ¹¹ CFU are mixed, wherein the lactic acid bacteria is Lactobacillus rhamnosus, Lactobacillus acidophilus, Lactobacillus delbruecki subsp. Bulgaricus is a mixed lactic acid bacterium mixed in a weight ratio of 1:0.5:0.5 to 1:2:2, and the rice bran-derived dietary fiber contains 20-30% by weight of insoluble dietary fiber and 2-10% by weight of soluble dietary fiber Iso using rice flour, characterized in that it is obtained by adding 100 to 200 parts by weight of water based on 100 parts by weight of rice bran, and then treating it with 0.005 to 0.1 parts by weight of cellulase and reacting for 1 to 3 hours so that it is 30 to 50 ° C. A method for producing a probiotic formulation containing maltooligosaccharide. According to claim 1,
A method for producing a probiotic formulation, characterized in that the isomaltooligosaccharide obtained in the third step has a sugar content of 16 to 18 Brix. According to claim 1,
The isomaltooligosaccharide obtained in the third step is isomaltose 30-40 g/L, pannose 8-15 g/L, isomaltotriose 15-25 g/L, isomaltotetraose (5-10 g/L) , containing 0.5 to 2 g/L of isomaltopentaose, and a method for producing a probiotic formulation, characterized in that the total isomaltooligosaccharide is 60 to 95 g/L. According to claim 1,
A method for producing a probiotic formulation, characterized in that the content of isomaltooligosaccharide is 50 to 60% by weight based on 100% by weight of the total saccharides in the composition containing isomaltooligosaccharide obtained in the third step. According to claim 1,
The method for producing a probiotic formulation, characterized in that the sugar content of the isomaltooligosaccharide obtained after concentration in the fourth step is 70-80 Brix. A probiotic formulation prepared by the method of claim 1 . A pharmaceutical composition for the prevention or treatment of irritable bowel syndrome, characterized in that it contains the probiotic agent of claim 6. A health functional food for the prevention or improvement of irritable bowel syndrome, characterized in that it contains the probiotic formulation of claim 6.

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