Patent Document

CROSS REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application is a national stage application of International Application PCT/KR2013/010180, filed Nov. 11, 2013, and claims the benefit of priority to Korean Patent Application No. 10-2012-0135779, filed Nov. 28, 2012, which is incorporated herein by reference in its entirety and for all purposes. 
     
    
     FIELD OF THE DISCLOSURE 
       [0002]    The present invention relates to a health supplement food containing  Pediococcus acidilactici  J9. 
       BACKGROUND 
       [0003]      Pediococcus acidilactici  produces bacteriocin (U.S. Pat. Nos. 5,260,212 and 5,175,252) and bacteriocin is known as a considerably widely used antibacterial agent. Yogurt containing bacteriocin is registered as U.S. Pat. No. 5,445,835. However, it was not easy to co-culture  Pediococcus acidilactici  with  Bacillus  bacteria. 
         [0004]    Lactic acid produced by Lactic acid bacteria is known to increase acidity (pH) in the intestine to enhance peristalsis of small intestine and thereby facilitate digestion and absorption, and to control enterokinesia in the large intestine and thereby prevent constipation and diarrhea. 
         [0005]    Yogurts and drinks produced by inoculating milk with Lactic acid bacteria, followed by fermentation and solidification are commercially available as fermented milk. 
         [0006]    Examples of Lactic acid bacteria used for production of yogurts include Lactic acid bacteria  bulgaricus, Streptococcus thermophilus, L. acidophilus  and the like. Distinct flavors are imparted to the fermented milk products by respectively culturing these Lactic acid bacteria at low temperatures and suitably mixing two or three thereof. These fermented milk products cannot survive at high temperatures and low pH and, as such, cannot reach human intestines. 
         [0007]    Commercially available grain-fermented enzyme-containing products are obtained by fermentation at 30° C. to 35° C. using a single fungus, so-called “ A. oryzae”.    
         [0008]    As a result of research and experiments at Konkuk University, Korea using  Pediococcus acidilactici  obtained from the University of Arkansas, US., it was found that bacteriosin which is a natural antibiotic substance produced by  Pediococcus acidilactici  destroys cell walls of  Listeria monocytogenes  causing sitotoxism and decreases the number of bacteria to 1.0×10 5  times after 6 hours. 
         [0009]    However, health supplement foods containing bacteriocin produced by  Pediococcus acidilactici  using grains have not been developed yet. 
       SUMMARY 
       [0010]    The present disclosure describes that control of pH is easy and food fermentation is thus further facilitated when  Pediococcus acidilactici  is co-cultured with  bacillus  and completed the present invention based on the discovery. Conventionally, co-culture of  Pediococcus acidilactici  with  bacillus  has been difficult. However, the present disclosure was completed based on the surprising discovery that  Pediococcus acidilactici  newly isolated by the named inventors was co-cultured well with  bacillus.    
         [0011]    Therefore, the described embodiments provide a solution to the above problems, and it is one object of the present embodiments to provide a food composition prepared by co-culturing  Pediococcus acidilactici  with  bacillus.    
         [0012]    It is another object of the present embodiments to provide a method for producing a health supplement food comprising co-culturing  Pediococcus acidilactici  with  bacillus.    
       Technical Solution 
       [0013]    In accordance with an aspect of the present disclosure, the above and other objects can be accomplished by the provision of a health supplement food produced by a method including preparing at least one Group A grain selected from the group consisting of brown rice and soybean, and at least one Group B grain selected from the group consisting of black rice, barley and sorghum, soaking a mixture of the Group A and B grains by immersing in water, cooking the soaked grains obtained by immersion using steam, cooling the cooked grains, adding previously cultured  Pediococcus acidilactici  J9 and the genus  bacillus  strains to the cooled grains, adding the resulting grains to a solid storage container and solid-culturing microorganisms at a temperature of 40 to 45° C. and at a humidity of 50 to 70% for 20 to 48 hours, further solid-culturing the solid culture product at a temperature of 40 to 45° C. and at a humidity of 20 to 40% for 30 to 60 hours, and drying the resulting solid culture product, followed by grinding. 
         [0014]    16S rRNA base sequences of the  Pediococcus acidilactici  J9 according to the present disclosure were analyzed in order to identify Lactic acid bacteria exhibiting superior heat resistance derived from a traditional Korean soybean paste (Cheonggukjang) by a molecular biological method. Homogeneity of the base sequence of selected strain was compared using the BLAST program of NCBI. As a result,  Pediococcus acidilactici  was identified at a homogeneity of 99% and this strain was called “ Pediococcus acidilactici  J9”. The  Pediococcus acidilactici  according to the present disclosure has antibacterial activity against tae which is a pathogen of bacteremia and tympanitis and is the most predominant pathogen of community-acquired pneumonia. Accordingly, the  Pediococcus acidilactici  J9 according to the present disclosure was deposited under an accession number of KCCM-11320P at the Korean Culture Center of Microorganisms. 
         [0015]    The genus  bacillus  bacteria may be  B. subtilis  or  B. pumilus . pH of the genus  bacillus  bacteria is maintained at a level suitable for culture of  Pediococcus acidilactici  J9 so that efficiency of fermentation was improved. There is no limitation as to the food and the food may be Doenjang or Cheonggukjang. 
         [0016]    Upper limits of the culture period, culture temperature and culture humidity are determined in consideration of cost and time required for culture of microorganisms and lower limits thereof are determined to satisfy sufficient conditions for obtaining culture period, culture degree and culture efficiency. 
         [0017]    The reason for using brown rice and soybean as major ingredients is to utilize pH control capability between  Pediococcus acidilactici  J9 (KCCM 11320P) and  Bacillus subtilis . Proliferation of Lactic acid bacteria causes formation of lactic acid as an organic acid from glucose, thus decreasing pH concentration and leading to acidification. However,  Bacillus subtilis  increases pH during fermentation to maintain pH at a neutral level of 6.0, thereby facilitating lactic acid bacteria. 
         [0018]    When grains are inoculated with Lactic acid bacteria,  Pediococcus acidilactici  J9, and fermented at high temperatures,  Listeria  bacteria inducing sitotoxism was not observed at all. After fermentation, Lactic acid bacteria is proliferated in grains and the number of bacteria cells reaches 1.2×10 9  CFU/g. Beneficial lactic acid bacteria function to suppress growth of harmful bacteria in the human intestine, strengthen intestinal peristalsis and thereby facilitate bowel movement. 
         [0019]    In accordance with another aspect of the presently described embodiments, provided is a method for producing a health supplement food, the method including preparing at least one Group A grain selected from the group consisting of brown rice and soybean, and at least one Group B grain selected from the group consisting of black rice, barley and sorghum, soaking a mixture of the Group A and B grains by immersing in water, cooking the soaked grains obtained by immersion using steam, cooling the cooked grains, adding previously cultured  Pediococcus acidilactici  J9 and the genus  bacillus  strains to the cooled grains, adding the resulting grains to a solid storage container and solid-culturing microorganisms at a temperature of 40 to 45° C. and at a humidity of 50 to 70% for 20 to 48 hours, further solid-culturing the solid culture product at a temperature of 40 to 45° C. and at a humidity of 20 to 40% for 30 to 60 hours, and drying the resulting solid culture product, followed by grinding. 
         [0020]    The genus  bacillus  bacteria may be  B. subtilis  or  B. pumilus . pH of the genus  bacillus  bacteria is maintained at a level suitable for culture of  Pediococcus acidilactici  J9 so that efficiency of fermentation is improved. There is no limitation as to the food and the food is Doenjang or Cheonggukjang. 
         [0021]    In accordance with another aspect of the presently described embodiments, provided is  Pediococcus acidilactici  J9 (KCCM 11320P) which is co-cultured well with  Bacillus  bacteria. As can be seen from Example 2 as described later,  Pediococcus acidilactici  J9 is a strain which is co-cultured well with  bacillus  and has superior pH stability and resistance to strong acid (see the following Example 3). 
       Technical Effects 
       [0022]    The food composition according to the presently described embodiments contains  Pediococcus acidilactici  and inhibits damage of grain fermented product by infection of microorganisms and thereby improves stability and lifespan of products. 
         [0023]    In accordance with the method for producing the food composition according to the presently described embodiments,  Pediococcus acidilactici  J9 is efficiently cultured due to control of pH by  bacillus  and production of bacteriocin is improved and stability and lifespan of the produced food composition are thus increased. 
         [0024]    As can be seen from the above, the food composition containing  Pediococcus acidilactici  J9 prevents contamination of other microorganisms due to proliferation at high temperatures during fermentation and glycolysis of microorganisms into grains is improved due to surface drying during fermentation. In addition, because of mutual pH buffering action between microorganisms of  Pediococcus acidilactici  J9 and the genus  bacillus , these microorganisms are efficiently proliferated. The food composition containing  Pediococcus acidilactici  J9 has the same functions as probiotics, for example, functions to facilitate intestinal peristalsis which is an inherent function of Lactic acid bacteria, the functions to suppress harmful bacteria and improve immunity. In addition, in accordance with recent research associated with  Pediococcus acidilactici  species in the academic world, bacteriocin and lactic acid as natural antibiotics created during fermentation of microorganisms are reported to exhibit antibacterial activity against  H. pylori  causing peptic ulcer such as stomach ulcer or duodenal ulcer and inhibitory activity to sitotoxism bacteria. The  Pediococcus acidilactici  according to the present invention exhibits inhibitory activity to  S. pneumoniae  as pneumonia bacteria. Accordingly, the food composition containing  Pediococcus acidilactici  J9 is expected to contribute to improvement of national health. 
     
    
     DETAILED DESCRIPTION 
       [0025]    Hereinafter, the presently described embodiments will be described in more detail with reference to examples. The scope to be protected by these embodiments is defined by claims and is not limited to the examples. The presently described embodiments can be implemented by those skilled in the art from the subject matters of the present disclosure and these various embodiments also fall within the right scope of the present inventors defined by claims. 
       Example 1 
       [0026]    Production of Food Using  Pediococcus acidilactici  J9 (KCCM 11320P) Stain 
         [0027]    A process for producing a grain-fermented enzyme-containing product by inoculating a  Pediococcus acidilactici  J9 (KCCM 11320P) strain is as follows. Grains used as major ingredients were 60% brown rice and soybean, and black rice, barley and sorghum were added such that the total weight of the grains was 200 kg, in order to improve flavor. The grains were soaked in water for 12 hours. After washing, the grains were cooked under steam in a cooker for about 40 minutes. The cooked grains were cooled.  Pediococcus acidilactici  J9,  B. subtilis  and  B. pumilus  were flask-cultured in an amount of 0.1% with respect to the total weight of grains. The number of the respective microorganisms was adjusted to 1.0×10 9  CFU/g. The microorganism culture solution was mixed with 2 L of distilled water and the cooked grains were inoculated with the mixture. The inoculated grains were stacked in a plastic box and were then solid-cultured in a fermentation device at a temperature of 42° C. and a humidity of 60% for 24 hours. Then, the inoculated crop was surface-dried at an inner temperature of 42° C. and a humidity of 40% or less for 48 hours to permeate microorganisms into the grains and thereby facilitate glycolysis. After surface drying, the inoculated grains were forcibly dried in a drier to cease fermentation. The dried grains were uniformly ground to 50 mesh in a grinding machine. 
       Example 2 
     Measurement of the Number of Proliferated  Pediococcus acidilactici  J9 and the Genus  Bacillus  Microorganism 
       [0028]    The number of  Pediococcus acidilactici  J9 was measured by counting yellow colonies after culturing at a dilution of 10 −7  with 9 mL of sterile physiological saline per 1 mg of a sample using a Plate count agar with BCP for measurement for 70 hours. The genus  Bacillus  microorganism was cultured in a nutrient broth agar medium in the same manner as above and white colonies were counted. The number of bacteria is shown in the following Table 1 below. 
         [0000]    
       
         
               
               
               
             
               
               
               
             
           
               
                   
                 TABLE 1 
               
               
                   
                   
               
               
                   
                   Pediococcus acidilactici  J9 
                 Genus  Bacillus   
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                 CFU/g 
                 1.2 × 10 9   
                 1.3 × 10 9   
               
               
                   
               
             
          
         
       
     
         [0029]    As can be seen from Table 1 above, microorganisms were proliferated well to about 10 9  per 1 g of the sample. In addition, during microorganism proliferation, formation of a natural antibiotic (Bacterocin, Pediosin) and an enzyme as secondary metabolite is well known in the art. 
       Example 3 
     Test of Antibacterial Activity of  Pediococcus  acidilactic 
       [0030]    The  Pediococcus  acidilactic was cultured and the concentration of bacteria was then measured using a quick start bovine serum albumin (BSA) standard kit (Bio-Rad, USA). As a result, the concentration of bacteria was 0.465 mg/ml. The culture solution was concentrated at 15,000 rpm for 5 minutes and the supernatant was collected. Then, the residue was filtered with a 0.2 μm filter (Milipore, USA) to remove bacteria and thereby prepare a  Pediococcus acidilactic  extract. 
         [0031]    Six standard strains shown in the following Table 2 were cultured using a standard flat comparison method in a CO 2  constant-temperature incubator at 37° C. for 24 hours. 
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
                 TABLE 2 
               
               
                   
                   
               
               
                   
                   
                 ATCC 
                   
                   
               
               
                   
                 Name 
                 number 
                 Note 
                 Grade 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 
                   Pseudomonas 
                 
                 27853 
                 MAC 
                 1 
               
               
                   
                 
                   aeruginosa 
                 
               
               
                 2 
                 
                   Staphylococcus 
                 
                 29213 
                 BAP 
                 2 
               
               
                   
                 
                   aureus 
                 
               
               
                 3 
                 
                   Streptococcus 
                 
                 49619 
                 BAP 
                 2 
               
               
                   
                 
                   pneumoniae 
                 
               
               
                   
               
             
          
         
       
     
         [0032]    A single colony-forming unit of each standard strain was seeded on a blood agar plate and a  P. acidilactici  extract was inoculated at 5 μl, 10 μl, 20 μl and 100 μl. The strains were dried in the air in a clean bench for 10 minutes and cultured in a CO 2  constant-temperature incubator at 37° C. for 24 hours. Then, diameter of the inhibition zone was measured by measuring the size of spots and results are shown in Table 3. 
         [0000]    
       
         
               
               
               
               
             
               
               
               
               
             
           
               
                   
                 TABLE 3 
               
               
                   
                   
               
               
                   
                 Name 
                 ATCC number 
                 Results 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 
                   Pseudomonas aeruginosa 
                 
                 27853 
                 No effect 
               
               
                 2 
                 
                   Staphylococcus aureus 
                 
                 29213 
                 No effect 
               
               
                 3 
                 
                   Streptococcus pneumoniae 
                 
                 49619 
                 effect 
               
               
                   
               
             
          
         
       
     
         [0033]    As can be seen from Table 3 above,  P. acidilactici  extract inhibited growth of  Streptococcus pneumoniae  ATCC49619. An inhibition diameter (mm) at different extract concentrations was not calculated for 5 μl, and inhibition diameters (mm) were 3 mm, 6 mm and 13 mm for different extract concentrations of 10 μl, 20 μl and 100 μl, respectively. Characteristic α-hemolysis of  Streptococcus pneumoniae  was not observed in a region where the  P. acidilactici  extract was inoculated and bacteria were not cultured, which means that growth of  S. pneumoniae  bacteria was inhibited. 
       Example 4 
     Test of pH Stability of  Pediococcus acidilactici  J9 
       [0034]    First, a 0.5% Lactobacilli MRS medium (Difco Laboratories Inc.) was diluted in 200 mL of distilled water and was sterilized and pH of the dilution was then adjusted to 3.0 with an HCl solution. 
         [0035]      Pediococcus acidilactici  J9 was diluted at a density of 3.0×10 8  CFU/g with physiological saline and was maintained for a lead time of 30 minutes. An aqueous solution (pH 3.0) was inoculated at 10% with the  Pediococcus acidilactici  J9 solution and at 37° C. and the number of lactic acid bacteria was measured after culturing for one hour and two hours. The number of  Pediococcus acidilactici  J9 was measured by counting yellow colonies after culturing at a dilution of 10 −7  with 9 mL of sterile physiological saline per 1 mg of a sample using a Plate count agar with BCP for measurement for 70 hours. 
         [0036]    As a result, it was confirmed that the number of bacteria was not varied at all. This means that  Pediococcus acidilactici  J9 had superior pH stability and resistance to strong acid. Accordingly, there was a high probability that  Pediococcus acidilactici  J9 could reach the human intestine alive when passed through the human stomach and intestine. 
         [0000]    
       
         
               
               
               
             
           
               
                   
                 TABLE 4 
               
               
                   
                   
               
               
                   
                 Acidity resistance test 
                   Pediococcus acidilactici  J9 
               
               
                   
                   
               
             
             
               
                   
                 Number of bacteria after one hour 
                 1.9 × 10 8   
               
               
                   
                 (CFU/g) 
               
               
                   
                 Number of bacteria after two hours 
                 1.2 × 10 8   
               
               
                   
                 (CFU/g) 
               
               
                   
                   
               
             
          
         
       
     
       Example 5 
     Test for Confirming Thermal Stability of  Pediococcus acidilactici  J9 
       [0037]    First,  Pediococcus acidilactici  J9 was cultured in a Lactobacilli MRS medium (Difco Laboratories Inc.) and immersed in 200 mL of distilled water in a constant temperature bath at 90° C. for 2 minutes. 
         [0038]    The number of  Pediococcus acidilactici  J9 was measured by counting yellow colonies after culturing at a dilution of 10 −7  with 9 mL of sterile physiological saline per 1 mg of a sample using a Plate count agar with BCP for measurement for 70 hours. 
         [0039]    As a result, regarding the number of bacteria,  Pediococcus acidilactici  J9, as a control group before test was 2.9×10 19  and the number of bacteria was 2.0×10 9  at 90° C. for 2 minutes, which means  Pediococcus acidilactici  J9 has superior thermal stability. 
         [0000]    
       
         
               
               
             
           
               
                 TABLE 5 
               
               
                   
               
               
                 Thermal stability test 
                   Pediococcus acidilactici  J9 
               
               
                   
               
             
             
               
                 Number of bacteria before test (CFU/g) 
                 2.9 × 10 10   
               
               
                 Number of bacteria at 90° C., after 2 
                 2.0 × 10 9    
               
               
                 minutes (CFU/g) 
               
               
                   
               
             
          
         
       
     
         [0040]    Although the preferred embodiments have been disclosed for illustrative purposes, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the disclosure as disclosed in the accompanying claims.

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