Description

LACTOBACILLUS PLANTARUM SE 1 USEFUL FOR MANUFACTURING LACTOBACILLUS CONTAINING BREAD

Technical Field

[1] The present invention relates to a lactobacillus plantaram SE 1 useful for manufacturing a lactic acid bacterium bread, and more particularly, to a lactobacillus plantarum SE 1 useful for manufacturing a lactic acid bacterium bread capable of improving a taste and flavor thereof and preventing a helicobacter pylori bacterium through a lactic acid bacterium derived from a soybean fermented bread. Background Art

[2] A lactic acid bacterium existed widely in the natural world is a microbe of generating an organic acid by using a carbohydrate such as a glucose or lactose. The lactic acid bacterium has been directly or indirectly used in a food from a long time ago. As a result of a research on an intestinal microflora of a man, it has been reported that the main lactic acid bacterium separated in the stomach of a healthy man is a Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus fermentum, Lactobacillus plantarum and so on. Such a lactic acid bacterium has a strong acid-proof and bile acid-proof. Presently, the lactic acid bacterium has been widely used as a microbial ag ent or vital cell preparation of a fermented goods and so on all over the world.

[3] It has been doubted that a helicobacter pylori bacterium causes various diseases in a stomach. Also, a population of about 50% is infected with the helicobacter pylori bacterium. Especially, it is presumed that an adult population of about 80% is infected with the helicobacter pylori bacterium in Korea and so forth. The helicobacter pylori bacterium can live in the stomach of a strong acid through the secretion of an enzyme of neutralizing the stomach acid. It has been known that the helicobacter pylori bacterium stimulates the gastric mucous membrane and causes an inflammation through the secretion of an enzyme of dissolving the stomach wells, thereby bringing about an inflammation of the stomach. Also, it is considered that the helicobacter pylori bacterium causes a stomach ulcer, a duodenal ulcer, a specific stomach cancer and so on. However, it has not been established how it causes the diseases. Since the diseases can be remarkably decreased through antibiotics against the bacterium, it generally cures the bacterium through the antibiotics.

[4] The helicobacter pylori bacterium as a gram-negative bacterium was firstly separated and identified from the mucous membrane tissue of a man by means of an Warren and Marshall in 1983. It was named as "helicobacter pylori" for his shape and the name of a pylorus living in the stomach.

[5] As described above, the helicobacter pylori bacterium can cause the stomach ulcer as well as the stomach cancer (Dubois, A. 1995, Spiral bacterium in the human stomach; the gastric Helicobacters. Emerg. Infect. Dis. 1:790085 ; Slomiany, B. L. and A. Slomiany, 1002. Mechanism of Helicobacter pylori pathogenesis ; focus on mucus, J. Clin. Gastroenterol. 14 Suppl 1 Sl 14-21). Also, where it is infected with the helicobacter pylori bacterium, it is continued for the decades. Moreover, since the helicobacter pylori bacterium is hardly naturally removed, it acts on the primary reason of a chronic inflammation of the stomach.

[6] In the meantime, it is established that the lactobacillus produces a lactate and an unknown material, so that it can prevent the helicobacter pylori bacterium. Up to present, it is known that the culture solution of a lactobacillus salivarius has a best effect on the growth prevention of the helicobacter pylori bacterium (Aiba, Y., N. Suzuki, A. M. Kabir, A. Takagi, and Y. Koga, 1998 Am. J. Gastroenterol. 93: 2097-2101). Also, it was confirmed that the lactobacillus can prevent the combination of a lewis antigen combined and attached to the mucous membrane of the helicobacter pylori bacterium (Lee, Y., E. Shin, J. Lee, and J.H. Park, 1999, Lactobacillus acidophilus inhibits the Helicobacter pylori adherence. J. Microb. Biotech, 9; 794-797).

[7] Meanwhile, a soybean has an abundant protein as well as a linoleic acid and a linolenic acid occupying 52% and 11% among the entire fatty acid respectively, so that it is a good source of a superior essential fatty acid. Also, it has been established that it has an isoflavon, a dietary fiber, a lecithin, a saponin and so on, it has an effect on a cancer, a fatness, and a control of a blood sugar and cholesterol. The protein of the soybean does not have a taste ingredient in itself. However, in the course of the inoculation and fermentation of a mildew or a bacterium, the protein of the soybean is dissolved by using a breakdown enzyme of the protein, thereby being flavored. Also, the soybean has much polysaccharides and the finished goods thereof has much water- soluble peptide and amino acid through the fermentation process using the microorganism, thereby being tasty and digesting well. The fermented soybean has residual products such as a salt and a lactic acid in comparison with non-fermented soybean products. The residual products play an important role in preventing the rot of the fermented product.

[8] During the inventors of the present invention product a soybean fermented material and develop various products using it, a bread including the soybean fermented material (referred as "fermented bread" herein below) has been prepared. Also, it has been found that the fermented bread itself contains a plenty of lactic acid bacterium. Accordingly, it has been filed with Korean Industrial Property Organization.

[9] The flavor, volume, taste, texture and so on affecting the quality of the fermented

bread using the soybean fermented material can be improved by means of the ingredient content of the soybean fermented material itself and the lactic acid bacterium of the soybean fermentation. It had been reported that the taste of the bread and the sensory quality (flavor) thereof are increased through a lactobacillus brevis and lactobacillus plantarum (KOREAN J. FOOD & NUTR. (1996) Vol. 9, No. 4, 509-515). Especially, there is reported that the lactobacillus plantarum serves to increase the functions of the anticancer and immunity as well as recently prevent the growth of the helicobacter pylori bacterium causing the stomach ulcer, duodenal ulcer and stomach cancer. In the Europe, the lactobacillus plantarum as a probiotic fungus(antibiotic in living body) is added to a yoghourt.

[10] During the inventors of the present invention are continuously researched on the soybean fermented material and the fermented bread, it is confirmed that the new microorganism is commonly existed in the soybean fermented material and the fermented bread and then, the inventive microorganism was separated and identified, thereby finding them.

Disclosure of Invention Technical Problem

[11] It is, therefore, an object of the present invention to provide a lactobacillus plantarum SE 1 fungus derived from a soybean fermented bread capable of improving a taste and flavor thereof and manufacturing a lactic acid bacterium bread.

[12] Another object of the present invention is to provide the lactic acid bacterium bread using the lactobacillus plantarum SE 1 fungus. Technical Solution

[13] To achieve the above objects of the present invention, there is provided a lactobacillus plantarum SE 1 derived from a soybean fermented bread useful for manufacturing a lactic acid bacterium bread (International deposit No. KCTC 10870BP).

[14] To achieve the above objects of the present invention, there is provided a soybean fermented material, wherein 5-7 wt % of lactobacillus plantarum SE 1 (International deposit No. KCTC 10870BP) is mixed with 5-12 wt % of a soybean powder passing through steam, cooling and powdering processes and 5-7 % of a sugar, and a water as a remainder and the mixture is fermented for 18-28 hours.

[15] Preferably, the soybean fermented material is a crude liquid of a liquid fermented material fermented in the type of a viscous yoghurt or a fermented powder of drying the crude liquid.

[16] To achieve the above objects of the present invention, there is provided a composition for baking, which contains 5-30 parts by weight of the soybean fermented material relative to 100 parts by weight of flour.

[17] To achieve the above objects of the present invention, there is provided a5. A dough for baking, which contains 5-30 parts by weight of the soybean fermented material relative to 100 parts by weight of flour.

[18] To achieve the above objects of the present invention, there is provided a lactic acid bacterium bread, wherein 5-30 parts by weight of the soybean fermented material relative to 100 parts by weight of flour is added and the additive is baked.

[19] Preferably, the lipid-based component as used herein may include, if not specifically limited to, a pseudo-ceramide, cholesterol and derivatives thereof, higher fatty acid, higher fatty alcohol, or any mixture thereof having the following formula 1 or 2. According to the present invention, the lipid-based component may include any lipid ingredient between horny cells.

Advantageous Effects

[20] As described above, according to the lactobacillus plantarum SE 1 useful for manufacturing a lactic acid bacterium bread according to the present invention, the lactic acid bacterium bread using the same has superior taste and flavor and the fermented soybean (soybean fermented material) can be prepared by using the fungus. Brief Description of the Drawings

[21] FIG. 1 is a photograph illustrating a colony of a lactobacillus plantarum SE 1 according to the present invention grown from a MRS agar medium including 1% of calcium carbonate (CaCo );

[22] FIG. 2 is a photograph illustrating a colony of a lactobacillus plantarum SE 1 according to the present invention grown from a MRS agar medium including 0.004% of bromocresol purple;

[23] FIG. 3 is a an optical microscope photograph illustrating of a lactobacillus plantarum SE 1 according to the present invention;

[24] FIG. 4 illustrates a photography for confirming a single band of the reported 419bp as a result of an agarose gel electrophoresis after the polymerase chain reaction (PCR) is carried by using a genome DNA and a colony of a separation fungus;

[25] FIG. 5 illustrates a growth curves of a lactobacillus plantarum SE 1 according to the present invention;

[26] FIG. 6 illustrates a ph values of a lactobacillus plantarum SE 1 according to the present invention; and

[27] FIG. 7 illustrates a sequencing as an identification result in that a genome DNA is separated from lactobacillus plantarum SE 1 and 16 rRNA sequencing is analyzed by means of an automated DNA sequencer so as to finally identify a fungus of a lactobacillus plantarum SE 1 according to the present invention. Best Mode for Carrying Out the Invention

[28] A preferred embodiment of the invention will be described in detail below.

[29] A lactobacillus plantaram SE 1 according to the present invention is separated from a fermented bread using a fermented soybean (soybean fermented material). The lactobacillus plantarum SE 1 is a new lactic acid bacterium such as a lactobacillus acidophilus, lactobacillus casei, lactobacillus fermentum, lactobacillus plantarum and so on separated in a stomach of a healthy man. It is deposited with a Korean Collection for Type Cultures attached to a Biological Resource Center of Korea Research Institute of Bioscience and Biotechnology (International deposit No. KCTC 10870BP).

[30] In the soybean fermented material according to the present invention, 5-7 wt % of lactobacillus plantarum SE 1 (International deposit No. KCTC 10870BP) is mixed with 5-12 wt % of a soybean powder passing through steam, cooling and powdering processes and 5-7 % of a sugar, and a water as a remainder and the mixture is fermented for 18-28 hours. Here, in case of using the soybean powder less than 5 wt %, there is a problem in that the nutritive ingredient content can be insufficient. On the contrary, in case of using the soybean powder more than 12 wt %, there is another problem in that the taste and flavor can be changed owing to the non-fermentation thereof. Also, in case of using the sugar less than 5 wt %, there is a problem in that the fermentability thereof can be lowered. On the contrary, in case of using the sugar more than 12 wt %, there is another problem in that a lot of sugar can be remained owing to the non-fermentation thereof. Moreover, in case of using the lactobacillus plantarum SE 1 less than 5 wt %, there is a problem in that the conversion rate of the effective ingredient from a protein to an amino acid can be lowered. On the contrary, in case of using the lactobacillus plantarum SE 1 more than 12 wt %, there is another problem in that the effective ingredients can be lost owing to the excessive fermentation thereof.

[31] In the present invention, a composition for baking containing 5-30 parts by weight of the soybean fermented material relative to 100 parts by weight of flour is provided. Here, the composition for baking can be prepared by mixing the flour as a main ingredient of bread with the soybean fermented material using the lactobacillus plantarum SE 1 according to the present invention. Also, the composition for baking is kneaded, molded and baked according to the conventional manner, so that it can prepare the lactic acid bacterium bread according to the present invention. That is, the composition for baking is kneaded in a water or milk and so on, and then, it is straight divided, molded and baked, thereby preparing the lactic acid bacterium bread according to the present invention.

[32] In the present invention, the dough for baking containing 5-30 parts by weight of the soybean fermented material relative to 100 parts by weight of flour is provided. Here, the dough is straight divided, molded and baked, so that it can prepare the lactic acid bacterium bread according to the present invention. The dough can be circulated

as it is. However, it is normally desirable that the frozen dough can be circulated or stored. The frozen dough is straight thawed, divided, molded and baked in the consumption spot, thereby preparing the lactic acid bacterium bread according to the present invention.

[33] Also, 5-30 parts by weight of the soybean fermented material relative to 100 parts by weight of flour is added thereto, thereby yielding the lactic acid bacterium bread by means of a general manner.

[34] Moreover, the lactic acid bacterium bread can be a crude liquid of a liquid fermented material fermented in the type of a viscous yoghurt or a fermented powder of drying the crude liquid. Here, in case of using the soybean fermented material less than the 5 parts by weight relative to 100 parts by weight of flour, there is a problem in that the taste and flavor can be lowered. On the contrary, in case of using the soybean fermented material more than the 30 parts by weight relative to 100 parts by weight of flour, there is another problem in that the peculiar shape and taste of the bread can be changed.

[35] Hereinafter, the present invention will be described in detail by way of the following examples and experimental examples, which are not intended to limit the scope of the present invention.

[36] Example 1

[37] Separation and identification of fungus

[38] A fermented bread using a soybean fermented material (lactic-acid fermented soybean, product name: soyghurt) provided by soyfarm co., ltd. is used as a sample. l,000g of a strong flour, 130g of a sugar, 30g of a powdered milk, 12g of a salt, 25Og of an egg, lOOg of a fresh cream, 300g of the soybean fermented material, and 150g of a butter are kneaded at a temperature of 27 ⁰ C to yield a dough. The yielded dough is first-fermented at a temperature of 28 ⁰ C and a humidity of 60% for about 60 minutes and is punched (beaten) to remove a gas, and then, is further fermented for 20 minutes. Then, it is divided and frozen at a temperature of -20 ⁰ C for one hour and then, refrigerated at a temperature of 1 ⁰ C for 9 hours. Thereafter, the refrigerated dough is second-fermented at a temperature of 33 ⁰ C and a humidity of 85% for about 40 minutes and then, baked according to a conventional method (for example, at an upper temperature of 200 ⁰ C and a lower temperature of 170 ⁰ C for about 11 minutes), thereby yielding the fermented soybean bread.

[39] Various beneficial bacterium including a microbe for secreting protein breakdown enzyme are observed in the fermented soybean bread (soyghurt). Only a lactic acid bacterium among them is selectively cultured. The results are shown in Table 1 below.

[40] Table 1

[41] [42] Separation of lactic acid bacterium from sample [43] A scrap of the fermented soybean bread is sufficiently soaked in 300ml of MRS fluid medium (note table 2 below on a medium composition) and cultured at a temperature of 37 ⁰ C for a night. Then, 100ml of culture fluid is smeared in a MRS agar medium including 1% of calcium carbonate (CaCo3) and cultured at a temperature of 37 ⁰ C for 48 hours. The colony having transparent rings is first-judged as the lactic acid bacterium. The colony is illustrated in FIG. 1. Also, 100ml of culture fluid is smeared in a MRS agar medium including 0.004% of bromocresol purple and cultured at a temperature of 37 ⁰ C for 48 minutes. The colony having yellow rings is second-judged as the lactic acid bacterium. The colony is illustrated in FIG. 2.

[44] Table 2

[45] [46] Identification of selected fungus [47] The identification on the lactobacillus plantarum SE 1 is carried through morphologically, physiologically, biochemically, and genetically experimental. The results are shown in Table 3 below and the optical microscope photograph is illustrated in FIG. 2.

[48] Table 3

[49] [50] The fermentation experimental of 49th-class on the fungus regarding as the lactic acid bacterium according to the present invention is carried using an API 50CHL kit (API bioMerieux, France). The results are shown in Table 4 below.

[51] Table 4

[52] [53] As seen from table 4, the SE 1 fungus is identified as the lactobacillus plantarum with a 99-percent reliability rating.

[54] According to a thesis of J.Microbiol.Biotechnol. (2000), 10(6), page 881-884, it has been disclosed in that the lactobacillus plantarum is searched as a product of the reaction after a polymerase chain reaction (PCR) is carried by using specific primers (note 13LP and 18LP in the thesis). Also, the specific primers has been designed and manufactured according to the thesis. Fig. 4 illustrates a photography for confirming a single band of the reported 419bp as a result of an agarose gel electrophoresis after the polymerase chain reaction (PCR) is carried by using a genome DNA and a colony of a separation fungus.

[55] [56] 16 rRNA sequencing [57] In order to finally identify the fungus, the genome DNA is separated from it and 16 rRNA sequencing is analyzed by means of an automated DNA sequencer. As a result, the lactobacillus plantarum is finally identified with the GenBank registration No. M58827 lactobacillus plantarum in a 99-percent reliability rating. The sequencing as the identification result is shown in FIG. 7.

[58] [59] Example 2 [60] Growth and pH change of lactobacillus plantarum SE 1 fungus according to temperature

[61] The lactobacillus plantarum SE 1 is inoculated into 100ml of MRS fluid medium with an amount of 107CFU/ml and cultured at temperatures of 30°c, 37°c and 42 ⁰ C for a night respectively. The growth curves of the fungus cultured by each temperature are illustrated in FIG. 5 and the ph values of the culture solution measured by each time are illustrated in FIG. 6. As shown in FIG. 5 and FIG. 6, the ph values are lower as time goes by. Also, after a lapse of five hours, the range of fluctuation in the pH is

small. Accordingly, it can be seen that the lactobacillus plantarum SE 1 creates an acid as a residual products of the culture solution. Moreover, after a lapse of fourteen hours, the cell proliferation is not active. Furthermore, it can be seen that the proper rearing temperature is about 30°c. Here, the growth at a temperature of 30°c through 42 ⁰ C similar to the temperature of the soybean fermentation means that the lactobacillus plantarum SE 1 can be considerably concerned in the soybean fermentation. Also, the creation of the acid means that the proliferation of other microbes can be decreased.

[62] [63] Example 3 [64] Acid-proof confirmation of lactobacillus plantarum SE 1 fungus [65] The lactobacillus plantarum SE 1 is inoculated into 10ml of MRS fluid medium with an amount of 107CFU/ml and cultured for a night. Only cells collected from the culture solution are inoculated into phosphate buffered saline of pH 4.0, pH 3.0 and pH 2,0 with an initial inoculation concentration of about 107CFU/ml. Then, they are fixed at a temperature 30°c for one, two and three hours respectively. Thereafter, ImI amounts picked out of them are diluted in a saline solution for dilution respectively. Then, 100ml of the diluted solution is smeared in a MRS agar plate medium and cultured at a temperature of 30 ⁰ C for 48 hours, thereby calculating and comparing the number of the cluster. The results are shown in Table 5 below.

[66] Table 5

[67] [68] Accordingly, it can be confirmed that the lactobacillus plantarum SE 1 can be survived even at a low pH. That is, it can be seen that the probability for existence thereof is highly in a stomach.

[69] [70] Example 4 [71] Bile acid-proof confirmation of lactobacillus plantarum SE 1 fungus [72] 100ml of the lactobacillus plantarum SE 1 is smeared in MRS solid mediums having 300mg/ml, 400mg/ml and 500mg/ml of a sterilized bile acid respectively and cultured at a temperature of 30 ⁰ C for 24 hours, thereby calculating and comparing the

number of the cluster. The viability of the lactic acid bacterium is shown in Table 6 below.

[73] Table 6

Concetration OD/D 300D/D 400D/D 500D/D

Number of 1.5*10 ⁸ 1.2*10 ⁸ 9.7*10 ⁷ 8.8*10 ⁷ bacteria

Unit: Number of bacteria (CFU/ml)

[74] As seen from Table 6, it can be found that the probability for existence thereof is highly in a stomach like the acid-proof thereof.

[75] [76] Example 5 [77] Preparation of soybean fermented material using lactobacillus plantarum SE 1 [78] 6 wt % of lactobacillus plantarum SE 1 (International deposit No. KCTC 10870BP) is mixed with 10 wt % of a soybean powder passing through steam, cooling and powdering processes, 6 % of a sugar, and a water as a remainder and the mixture is fermented for 24 hours, thereby yielding a soybean fermented material. In the fermentation of the soybean, it seems that the protein of the soybean is resolved through the operation of a bacillus subtilis existed in the soybean. Also, it is confirmed that a lactic acid bacterium content of the finally yielded soybean fermented material is 520,000,00OCFUVmI. Accordingly, it can be confirmed that the soybean fermented material having very high lactic acid bacterium content can be prepared by using the lactobacillus plantarum SE 1 according to the present invention in comparison with the conventional lactic acid bacterium content of 400,000,000CFU/ml using the conventional lactic acid bacterium such as the soyghurt.

[79] [80] Example 6 [81] Preparation of bread containing lactobacillus plantarum SE 1 and taste examination therof

[82] A lactic acid bacterium bread is prepared according to a prescription of Table 7 by using the soybean fermented material yielded by the example 5. [83] 15 parts by weight of the soybean fermented material relative to 100 parts by weight of flour is added thereto, thereby yielding the lactic acid bacterium bread by means of a general manner. Here, it is confirmed that a lactic acid bacterium content of the finally yielded lactic acid bacterium bread is 450CFU/ml. Accordingly, it can be confirmed that the lactic acid bacterium bread having very high lactic acid bacterium content can be prepared by using the lactobacillus plantarum SE 1 according to the

present invention in comparison with the conventional lactic acid bacterium content of 360CFU/ml using the conventional lactic acid bacterium such as the soyghurt.

[84] Also, in the valuation of taste, the lactic acid bacterium bread and the general bread are applied to the sensory quality test through valuers of 50 persons. Also, five points measuring manners (5: excellent, 4: good, 3: normal, 2: bad, 1: very bad) is used as the test method. The results are shown in Table 8 below.

[85] Table 7

[86] Table 8

[87] [88] The majority of the valures judged that the lactic acid bacterium bread according to the present invention is destitute of specific smell, pleasant, and soft and. Accordingly, there are effects in that the lactic acid bacterium bread according to the present

invention can be prepared by the fungus and the plenty of the beneficial lactic acid bacterium can be easily took with the bread.

[89] Also, the lactobacillus plantarum lactic acid bacterium living in the stomach of an animal is a typical microorganism beneficial to its body. Moreover, the lactobacillus plantarum as the probiotic lactic acid bacterium is widely used in the fermented milk or the vital cell preparation. Accordingly, there are effects in that it tastes good and the lactic acid bacterium bread can be took in place of a rice or meal, thereby the bread and the beneficial lactic acid bacterium can be took together and it can effectively prevent the rearing of the harm microorganism.

[90]

Industrial Applicability

[91] The present invention relates to a lactobacillus plantarum SE 1 useful for manufacturing a lactic acid bacterium bread capable of improving a taste and flavor thereof and preventing a helicobacter pylori bacterium through a lactic acid bacterium derived from a soybean fermented bread.