KR20150040842A - Method for Preparing Galactooligosaccharide Using Lactobacillus Extract and Whey - Google Patents

Abstract

The present invention provides a method for preparing novel galactooligosaccharides, wherein the method comprises incubating lactobacillus in a whey medium for lactobacillus cultures and preparing galactooligosaccharides by applying the incubated lactobacillus to a whey medium for producing galactooligosaccharides. The present invention provides the galatooligosaccharides that can be consumed along with probiotics. According to the invention, production costs can be drastically reduced compared with that of existing production processes.

Description

Technical Field [0001] The present invention relates to a method for preparing galactooligosaccharide using Lactobacillus extract and whey,

The present invention relates to a method for producing galactooligosaccharides using lactic acid bacterium extract and whey.

Lactose, the main carbohydrate of milk and dairy products, is a disaccharide composed of glucose and galactose, contained only in the milk of all mammals and contains 4-5 g of lactose per 100 g of milk. However, lactose is a nutritional problem for most African black and Asian yellow adults. Lactose intolerance, which causes symptoms such as abdominal distension, abdominal pain, convulsions and diarrhea, which is caused by insufficient degradation of lactose in the small intestine due to insufficient degradation of lactose, Causing problems such as reduced consumption and quality deterioration in the manufacture of dairy products (Jang et al ., Korean J. Food Sci . Technol ., 22 (4), 426-433 (1990)).

Whey is an opaque, yellowish-green liquid that is released from the curd during cheese production. As the consumption of cheese has increased recently, production has also increased and it is a byproduct by-product that is recognized as an important substitute for skim milk. The average amount of whey produced when producing 1 kg of cheese is 9 kg, 9 times the cheese yield (Yoon et al., Korean J. Food Sci, Ani. Resour ., 17 (1), 38-44 (1997)).

Lactic acid bacteria are well known as " probiotics " which inhibit the growth of harmful bacteria by releasing lactic acid and lowering intestinal pH as lactic acid bacteria grow in the small intestine. Lactobacillus The probiotic acidophilus is found first, after studies have been actively conducted on the efficacy of different probiotic Lactobacillus species. Lactic acid bacteria also inhibited lactose intolerance (Mcdonough et al ., Am . J. Clin . Nutr ., 45, 570-574 (1987)) and the role of lowering cholesterol (Holzapfel et al ., Int J Food Microbiol . 26, 41 (2): 85-101 (1998)) and reduction of the risk of colorectal cancer (Sanders, Int . Dairy J. , 8, 341-347 (1998)).

 As the level of living has improved, interest in health has increased, and domestic food companies are also making efforts to develop safe natural food materials. As a result of these efforts, various oligosaccharide products have been developed and marketed. Oligosaccharides are processed sugar solutions obtained from carbohydrate raw materials. The oligosaccharides currently produced include fructooligosaccharide, maltooligosaccharide, isomaltooligosaccharide, and galactooligosaccharide (GOS). Fructo-oligosaccharides are liquid or powdery forms obtained by filtration, purification and concentration of a sugar solution obtained by using an enzyme to bind one or more fructose molecules or an sugar solution obtained by using an enzyme from a saccharide raw material. Isomaltooligosaccharide is a glucose Refers to a liquid or powdered liquid obtained by filtering, purifying, and concentrating a sugar solution obtained by allowing an enzyme to act on a molecule so that the molecule has a basic structure of branching bonding. Galacto-oligosaccharide refers to a liquid or powder form of a transformed galacto-oligosaccharide solution obtained by the action of an enzyme using a carbohydrate raw material, or a sugar solution of raffinose or starch extracted from sugar beet or soybean, filtered, purified and concentrated. Refers to a liquid or powdery product obtained by processing a sugar solution obtained by allowing an enzyme to act so that 3-10 glucose molecules are linearly linked by using a saccharide raw material (Food Science, 2009).

These oligosaccharides are not degraded in the small intestine but go to the large intestine and act as growth factors of Bifidobacteria , a useful bacterium in the intestines, to inhibit the growth of intestinal E. coli, enterococci and pernicious bacteria. In addition, it is useful for people who are restricted in sugar consumption due to diabetes and the like, because they exhibit the effects of synthesis of nutrients, promotion of metabolism, promotion of immune function and improvement of constipation by promoting intestinal motility. In addition, it is also used as a food material such as formula milk, beverage, ice cream, and bread, medicine, and livestock feed (Kim CR, Korean J. Food Nutr ., 12 (1), 50-54 (1999)).

The function of oligosaccharides as a growth factor of Bifidobacterium bacterium, which is beneficial in the intestine, has been highlighted, and β-galactosidase (EC 3.2) has been used for the long time for hydrolysis of lactose to produce galacto-oligosaccharides. 1.23) by transgalactosidation / transgalactosylation (In et al ., Korean J. Food Sci . Technol ., 29 (2), 376-378 (1997)). In particular, galactooligosaccharide is abundant in the components of breast milk and is an indigestible substance, and is currently widely used as an additive for dairy products in the food industry. Beta-galactosidase, an enzyme that produces such galacto-oligosaccharides, is also known as lactase and is produced by a wide variety of microorganisms such as bacteria, yeast, and fungi. Bacillus circulans (Fujimoto et al . , Glycoconjugate Journal , 15, 155-160 (1998)), Bacillus megaerium (Li et al . , Appl Biochem Biotechnol , 158, 192-199 (2009)), Bifidobacterium adolescentis (Hinz et al . , Appl . Microbiol . Biotechnol . , 66, 276-284 (2004)), Bifidobacterium infantis (Hung et al . , Appl . Microbiol . Biotechnol ., 58, 439-445 (2002)), Enterobacter agglomerans (Lu et al . , J. Agric . Food Chem . 54, 4989-4998 (2007)), Lactobacillus reuteri (Nguyen et al . , FEMS Microbiol Lett ., 269, 136-144 (2007)), Streptococcus pneumonia (Jeong et al . , J. Bacteriol ., 191 (9), 3011-3023 (2009)). Yeasts include Cryptococcus laurentii (Ohtsuka et < RTI ID = 0.0 > al ., J. Ferment . Bioeng ., 70 (5), 301-307 (1990)), Kluyveromyces fragilis (Ladero et al ., Enzyme Micro . Technol ., 30, 392-405 (2002)), Kluyveromyces lacits (Kim et al ., Biotechnol . Lett ., 25, 1769-1774 (2003)), Sterigmatomyces elviae (Onishi and Tanaka, Appl Environ Microbiol . Nov; 61 (11): 4026-30 (1995)). Fungi include Aspergillus aculeatus (van Casteren et al ., Carbohydr . Res ., 329, 75-85 (2000)), Aspergillus oryzae (Todorova-Balvay et al ., J. Mol . Recognit ., 19, 299-304 (2006)), Bullera singularis (Cho et al ., Biotechnol . Lett ., 25, 2107-2111 (2003)), Sporobolomyces singularis were studied (Table 1-2).

Biochemical Characteristics of Microorganism Beta-Galactosidase Having Antigenic Activity Temperature rating Enzyme-derived system Temperature
(° C) pH activation
(U mg ^-1 ) Reference Cold air
(Psychrophile) Arthrobacter
psychrolactophilus 10 8.0 20 Nakagawa et al . 2007 Mesophile
(Mesophiles) Aspergillus aculeatus 55-60 5.4 24 van Casteren et al . 2000 Aspergillus oryzae 30 4.8 40 Todorova-Balvay et al . 2006 Bacillus circulans 50 5.0-6.0 5.1 Fujimoto et al . 1998 Bacillus megaterium 40 6.0-9.0 60 Li et al . 2009 Bifidobacterium
 adolescentis 50 6.0 526 Hinz et al . 2004 Bifidobacterium
 bifidum 37 6.5 - Dumortier et al . 1994 Bifidobacterium
 infantis 50-60 7.5 569 Hung and Lee 2002 Bullera singularis 50 5.0 56 Cho et al . 2003 Cryptococcus laurentii 58 4.3 12 Ohtsuka et al . 1990 Enterobacter
 agglomerans 37-40 7.5-8.0 - Lu et al . 2007 Kluyveromyces fragile 30 6.5 - Ladero et al . 2002 Kluyveromyces lactis 37-40 6.6-7.0 - Kim et al . 2003 Lactobacillus
acidophils 55 6.5-8.0 230 Nguyen et al . 2007 Lactobacillus reuteri 50 6.0-8.0 180 Nguyen et al . 2006 Sporobolomyces
 singularis 30 4.0 8.7 Ishikawa et al . 2005 Sterigmatomyces
 elviae 85 4.5-5.0 20 Onishi and Tanaka 1995 Sterptococcus
 pneumonia 30 5.5-7.5 - Jeong et al . 2009 Alicyclobacillus
 acidocaldarius 65 5.5 592 Di Lauro et al . 2008 Bacillus
stearothermophilus 70 7.0 125 Chen et al . 2008 Caldicellulosiruptor
 saccharolyticus 80 6.0 211 Park and Oh 2009 Geobacillus
 stearothermophilus 65 6.5 0.5 Placier et al . 2009 Saccharopolyspora
 rectivirgula 60 6.5-7.2 - Nakao et al . 1994 Thermus sp . 70 6.5 - Ladero et al . 2002 Thermus aquaticus 80 5.5 5.7 Berger et al . 1997 Thermophile
(Hyperthermophiles) Sulfolobus solfataricus 75 6.5 116 Pisani et al . 1990 Thermotoga maritima 80-85 6.5 70 Kim et al . 2004

Park and Oh, 2010

Production of galacto-oligosaccharides from galactose using microbial beta-galactosidase Biocatalyst Enzyme derived Temperature
(° C) pH Lactose
(g L ^-1 ) GOS ^a ,%
(Conc, g L ^-1 ) productivity
(g L ^-1 h ^-1 ) Reference Enzyme
(Crude
enzymes) Aspergillus oryzae 40 4.5 380 31 24 Iwasaki et al . 1996 Bacillus circulans 40 6.0 46 24 2.2 Mozaffar et al . 1985 Bifidobacterium
 longum 45 6.8 400 33 13 Hsu et al . 2007 Geobacillus
 stearothermophilus 37 6.5 180 2 [2] 0.4 Placier et al . 2009 Lactobacillus reuteri 30 65 205 38 3.9 Splechtna et al . 2006 Penicillium sp . 55 4.0 400 40 [160] - In and Chae 1998 Talaromyces
 thermophilus 40 6.5 200 50 [100] 13 Nakkharat et al . 2006 Purified enzyme
(Purified
enzymes) Bullera singularis 50 5.0 180 50 [90] 3.9 Cho meat al . 2003 Enterobacter
 agglomerans 50 7.5 125 38 [47] 3.9 Lu et al . 2007 Lactobacillus
acidophils 30 6.5 205 39 7.9 Nguyen et al . 2007 Penicillium
 simplicissiumum 50 6.5 600 31 [183] 11 Cruz et al . 1999 Saccharopolyspora
 rectivirgula 60 6.5 600 44 [264] - Nakao et al . 1994 Sterigmatomyces
 elviae 60 5.0 200 39 [78] 3.2 Onishi and Tanaka
1995 Recombinant enzyme
(Recombinant
enzymes) Bifidobacterium
 infantis 60 7.5 300 63 [190] 13 Hung and Lee 2002 Pyrococcus
 furiosus 80 5.0 270 22 [60] - Bruins et al . 2003 Sulfolobus solfataricus 80 6.0 600 53 [315] 5.6 Park et al . 2008 Thermotoga maritima 80 6.0 500 19 [97] 18 Ji et al . 2005 Thermus sp . 70 7.0 300 30 [91] - Akiyama et al. 2001 Geobacillus
 stearothermophilus
 R109W 37 6.5 180 23 [41] 6.9 Placier et al . 2009 Toluene-treated cells
(Toluene-treated cell) Bifidobacterium
 bifidum 39 6.8 500 20 [99] 65 Tzortzis et al . 1996 Rhodotorula minuta 60 6.0 200 38 [76] 3.2 Onishi and Yokizeki
1996 Sirobasidium magnum 50 - 360 38 [136] 3.2 Onishi et al . 1996 Sirobasidium magnum 50 - 360 67 [242] 5.8 Onishi et al . 1996 Immobilized enzyme
(Immobilized
enzymes) Aspergillus candidus 40 6.5 400 37 [148] 87 Zheng et al . 2006 Aspergillus oryzae 40 4.5 400 27 106 Albayrak and Yang
2002 Bacillus circulans 40 6.0 46 40 4.2 Mozaffar et al . 1986 Bacillus circulans 45 3.7 300 54 [120] 4.8 Shin and Yang 1998 Kluyveromyces lactis 40 7.0 400 25 [99] 25 Chockchaisawasdee et al . 2004 Talaromyces
 thermophilus 40 6.5 200 50 70 Nakagawa et a l. 2006 Immobilized cell
(Immobilized
cells) Cryptococcus laurentii 40 4.3 200 40 [80] 3.6 Ozawa et al . 1989 Sporobolomyces
 singularis 55 5.0 600 40 [242] 8.7 Sakai et al . 2008

Park and Oh, 2010

Enzymatically called beta-galactosidase, which is called lactase, is an enzyme that hydrolyzes the β (1 → 4) bond of lactose and breaks down into glucose and galactose. It reacts with galactose, which is cut from lactose, to other lactose Is an enzyme that produces galactooligosaccharides (Park et < RTI ID = 0.0 > al ., Appl . Microbiol . Biotechnol ., 85, 1279-1286 (2010)).

Water-soluble enzymes are generally unstable in their enzymatic activity, and it is difficult to separate the products and enzymes after the reaction, so that expensive enzymes can not be reused and the reaction process is not continuous. As a solution to this problem, the enzyme immobilization has been recognized as a very useful method, and research on this has been actively conducted (Ob et al ., Biochem . Educ ., 28, 164-168 (2000)), (Tanaka et al ., J. Biochem ., 77 (1): 241-247 (1975)), (Squillante et al ., J. Microencapsul ., 20 (2), 153-167 (2003)), (Nakkharat et al ., World J. Microbiol . Biotechnol ., 23 (6), 759-764 (2006)).

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have sought to develop a method for producing galactooligosaccharide at low cost using a whey culture medium. As a result, it is possible to produce lactic acid bacteria in a whey culture medium for lactic acid bacteria culture; And a step of producing the galactooligosaccharide by applying the lactic acid bacterium to a whey culture medium for producing galactooligosaccharide. According to such a process, the lactic acid bacteria cultured in the whey culture medium can be applied to the whey culture medium for the production of galactooligosaccharide to produce the galactooligosaccharide continuously without further purification, thereby significantly reducing the production cost compared with the conventional production process Thereby confirming the completion of the present invention.

Accordingly, it is an object of the present invention to provide a method for producing galactooligosaccharide which is simpler in comparison with the conventional production process using whey culture medium, and significantly reduces the production cost.

Another object of the present invention is to provide a galactooligosaccharide produced using a whey culture medium.

According to one aspect of the present invention, the present invention provides a method for preparing galactooligosaccharides using a whey culture comprising the steps of:

(a) culturing a lactic acid bacterium in a whey culture medium for culturing a lactic acid bacterium; And

(b) applying the lactic acid bacterium to a whey culture medium for producing galactooligosaccharide to produce galactooligosaccharide.

According to one aspect of the present invention, there is provided a method for producing lactic acid bacteria, comprising the steps of: (a) culturing a lactic acid bacterium in a whey culture medium for culturing a lactic acid bacterium comprising 10 to 15 wt% of a whey powder and 0.5 to 2 wt% And (b) applying the lactic acid bacterium to a whey culture medium for producing galactooligosaccharide containing 20 to 40% by weight of a whey powder to produce a galactooligosaccharide.

The present inventors have sought to develop a method for producing galactooligosaccharides that significantly reduces the time and cost of production compared to the conventional production process using whey media. The present inventors have cultivated lactic acid bacteria in a whey culture medium for lactic acid bacteria culture to obtain lactic acid bacteria and applied them to a whey culture medium for the production of galactooligosaccharide to produce a novel galacto-oligosaccharide producing method. Conventionally, lactate production required for the production of galactooligosaccharide was carried out in fungi or yeast. In order to prevent infestation of fungi or yeast in vivo, it was necessary to remove the fungus or yeast and purify lactase in the final step.

In the present invention, since lactase is produced from a lactic acid bacterium which is a probiotic useful for living bodies, it can be used for the production of galactooligosaccharide without a separate purification process. Therefore, a series of processes from the cultivation of lactic acid bacteria containing lactase to the production of galactooligosaccharide can be carried out continuously without a separate purification step, thereby saving time and cost.

 The present inventors have found that culturing lactic acid bacteria in a whey culture medium for lactic acid bacteria culture; And a step of applying the lactic acid bacteria to a whey culture medium for producing galactooligosaccharide to produce a galacto-oligosaccharide. According to such a process, a lactic acid-containing lactic acid bacterium is applied to a whey culture medium It was confirmed that galactooligosaccharide, which is a growth factor of beneficial bacteria in the intestine, can be produced by simultaneously producing lactic acid bacteria and galactooligosaccharide which are beneficial to the living body.

The method of the present invention will be described in detail in each step as follows:

Step (a): for culture of lactic acid bacteria In whey medium  Cultivation of lactic acid bacteria

According to the present invention, a whey culture medium for lactic acid bacteria culture is prepared prior to culture of lactic acid bacteria.

As used herein, the term " lactic acid bacteria " refers to a group of bacteria that produce lactic acid as a major product of carbohydrate metabolism. The lactic acid bacteria in the present invention can be selected from lactic acid bacteria known in the art, for example, Lactobacillus paracasei paracasei , Lactobacillus rhamnosus , Lactobacillus brevis , Lactobacillus < RTI ID = 0.0 > confusus , Lactobacillus reuteri , Lactobacillus < RTI ID = 0.0 > delbrueckii , Lactobacillus < RTI ID = 0.0 > acidophilus , Lactobacillus plantarum , Lactococcus lactis, lactis , Lactobacillus < RTI ID = 0.0 > helveticus or Leuconostoc mesenteriodes , preferably Lactobacillus paracasei, Lactobacillus helveticus, Lactobacillus acidophilus or Lactobacillus lutei, more preferably Lactobacillus paracasei, to be.

The whey culture medium for culturing a lactic acid bacteria used in the present invention includes a carbon source, a nitrogen source and a whey ingredient. In the culture medium for culturing a lactic acid bacteria of the present invention, various carbohydrates can be used as the carbon source. Preferably, the carbohydrate is selected from the group consisting of glucose (glucose), sucrose (sucrose), maltose (maltose), fructose (fructose), lactose And more preferably sucrose, fructose, glucose, galactose, arabinose or lactose, and most preferably glucose.

In the culture medium for cultivation of lactic acid bacteria of the present invention, a variety of organic nitrogen sources may be used as the nitrogen source, preferably yeast extract, soytone, peptone, beef extract, tryptone or casitone, more preferably yeast extract, peptone, tryptone or soytone, and most preferably yeast extract. When the yeast extract is used as the nitrogen source, its content is preferably 0.3-3%, more preferably 0.5-2%, and most preferably 0.75-1.2%.

The whey of the whey culture medium for lactic acid bacteria cultivation may be used as a liquid remaining after coagulation of the milk component from the mammal, which is sterilized in a liquid state and used as it is or may be pulverized by freeze drying or the like. Whey can be purchased directly from animals or purchased commercially. Such whey may be, but is not limited to, milk, goat milk, goat milk, camel milk. The whey used in the present invention is preferably a powdered whey.

According to a preferred embodiment of the present invention, the whey culture medium for lactic acid bacteria is prepared by mixing distilled water into powdered whey, adding alcalase, completely dissolving in a constant temperature water bath, adding yeast extract and ammonium citrate, do.

 The powdery whey added to the whey culture medium for lactic acid bacteria culture is 5-20% by weight, preferably 5-15% by weight, more preferably 8-16% by weight, and most preferably 9-12% by weight .

The lactic acid bacteria culture can be carried out through various methods known in the art.

The inoculation of the lactic acid bacteria is carried out by adding the lactic acid bacteria pre-cultured to the culture medium in an appropriate amount so that the lactic acid bacteria can sufficiently proliferate under the culture conditions. In the present invention, the culture medium which can be used for the lactic acid bacteria pre-culture includes any medium used for culturing the lactic acid bacteria strain, but preferably a culture medium such as MRS (deMan Rogosa Sharpe) liquid medium, APT (All Purpose with Tween) medium or BHI ) Medium, and most preferably it is an MRS liquid medium.

The cultivation of lactic acid bacteria for the production of lactic acid bacteria enzyme solution can be carried out by adding 1-5% (v / v) of the pre-cultured lactic acid bacteria culture to the lactic acid bacteria culture whey culture medium. The cultivation of the lactic acid bacterium can be carried out according to the culture conditions of the lactic acid bacteria known in the art. Such a process can be easily adjusted according to the strain selected by a person skilled in the art (James et al., Biochemical Engineering , Prentice-Hall International Editions). The cells can be suspended or adhered according to the cell growth method, and can be carried out in a batch, fed-batch or continuous-feeding manner depending on the culture method.

According to a preferred embodiment of the present invention, the activity of the lactic acid bacteria is inoculated into the MRS liquid medium and cultured to inoculate the lactic acid bacteria in the whey culture medium. In the present invention, the lactic acid bacteria culture temperature is preferably 20-35 캜, more preferably 25-32 캜, and most preferably 28-32 캜.

The step (a) is as follows: The lactic acid bacteria are inoculated in 10 mL of the MRS liquid medium, pre-cultured at 30 DEG C for 18 hours, and cultured in a whey culture medium 100 for lactic acid bacteria cultivation mL, and cultured at 30 ° C for 18 hours.

Step (b): Using lactic acid bacteria Galacto-oligosaccharide  production

The lactic acid bacteria are applied to a whey culture medium for producing galactooligosaccharide to produce galactooligosaccharide.

In the present invention, galactooligosaccharide means a generic term of oligosaccharide produced by reacting lactase with lactose. In one embodiment, the galactooligosaccharide is represented by the general formula Gal- (Gal) n -Glc (wherein Gal represents galactose residue, Glc represents glucose residue, and n represents an integer of 1 to 4) Preferably, the galactooligosaccharide is selected from the group consisting of 4'-galactosyllactose in which galactose is linked to beta-1,4 bonds to lactose, and beta-1,4-linkage in which galactose is bonded to 4'- And at least one member selected from the group consisting of 4-saccharides to 6-saccharides and transition 2-sugars.

The 4'-galactosyl lactose BP gambling compared to raffinose or rakchyuroseu Te Solarium Al Dore sentiseu (Bifidobacterium aldolescentis), Bifidobacterium Bifidobacterium bonus (Bifidobacterium bifidum ) or Bifidobacterium ( Bifidobacterium < / RTI > breve) are available only by Bifidobacterium bacteria, etc., and other intestinal harmful bacteria are not able to use almost, is reported to be an advantage to help maintain the balance of intestinal lactic acid bacteria and (Ohtsuka K. et al., Bifidus Vol 2 pp. 143-149 (1989)) and improve the bioavailability of iron (D. Perez-Conesa et al., Food Science and Technology International , Vol. 13, pp. 69-77 (2007)). In addition, Streptococcus mutans , a causative organism of cavity, mutans and the like, and insoluble glucan is not produced, which is advantageous in that it is characterized by low cavities (S. Hata. et al., Oral Microbiology and Immunology , Vol 16 pp. 57-62 (2001)), 4 ' -galactosyllactose, a representative oligosaccharide of breast milk, is essential as a constituent of infant formula to reproduce the metabolic enhancement function of breast milk.

The powdery whey added to the whey medium for production of galactooligosaccharide used in the present invention is 20-45 wt%, preferably 25-40 wt%, more preferably 30-40 wt%, and most preferably, 30-35% by weight.

In the present invention, the step (b) of producing the galactooligosaccharide can be carried out within a temperature and a culture time range at which the enzyme is not inactivated. Preferably, the step (b) Lt; / RTI > The step (b) may be carried out preferably for 2-8 hours, more preferably 3-6 hours, most preferably 4-5 hours. The step (b) may be carried out at a temperature of preferably 20 to 40 캜, more preferably 25 to 35 캜, and most preferably 29 to 32 캜.

According to a preferred embodiment of the present invention, a step of obtaining a lactic acid bacteria-containing enzyme solution containing a lactase from the culture medium of lactic acid bacteria obtained in step (a) may be further included between steps (a) and (b). In detail, the lactic acid bacteria in the culture broth can be recovered and subjected to centrifugation or filtration to separate the culture medium, and these steps can be carried out according to the needs of a person skilled in the art. The obtained lactic acid bacterium can be preserved by freezing or freeze-drying according to a conventional method so as not to lose its activity. For example, it may be stored at -80 ° C, including glycerol, or lyophilized in sterile 10% skimmed milk. In addition, the microorganism according to the present invention can be improved or improved so as to have an activity equivalent to or higher than that by a conventional physicochemical mutation method or the like known in the art.

The lactic acid bacterial enzyme solution used in the present invention is a solution obtained by crushing the lactic acid bacteria obtained by centrifuging the culture solution of the lactic acid bacteria.

According to the present invention, the lactic acid bacteria enzyme solution includes lactase. The lactase is an enzyme that hydrolyzes the beta-galactoside bond and is also referred to as beta-galactosidase.

In the present invention, the lactase reacts with lactose having a beta-galactoside bond as a substrate to hydrolyze it with galactose and glucose, and at the same time, conducts galactooligosaccharide formation reaction by transferring galactose to lactose. The lactase also produces a galactose enzyme intermediate product containing a galactose residue from a lactose substrate and produces a different product (i.e., a different type of galactooligosaccharide) depending on the substance (galactose receptor) that contains the galactose residue of the galactose enzyme intermediate As the reaction proceeds, the reaction product including the resulting galactooligosaccharide is further decomposed and recombined to produce various products.

The lactic acid bacteria enzyme solution used in the present invention may be immobilized on a carrier for immobilization. Preferably, the carrier for calibrating the lactic acid bacteria enzyme solution is selected from the group consisting of cellulose, dextran, agarose, polyacrylamide, sodium alginate, ceramic, slag, slag or plastics, more preferably cellulose, dextran, agarose, sodium alginate, ceramic, and most preferably sodium alginate.

In the present invention, the soda alginate used for immobilizing the lactic acid bacteria enzyme solution is preferably 0.5-3.0 wt%, more preferably 1.0-2.0 wt%, and most preferably 1.3-1.6 wt%.

The method of immobilizing the lactic acid bacteria crude enzyme solution of the present invention can be carried out by various methods known in the art, such as covalent bonding, ionic bonding, physical adsorption or biochemical bonding.

In the present invention, the step of preparing the lactic acid bacteria enzyme solution may be carried out within a pH range at which the enzyme is not inactivated, preferably at an optimal pH range of the enzyme to be used. This step is preferably carried out at pH 6-8, more preferably at pH 6.0-7.5, most preferably at pH 6.5-7.0.

The above steps will be described with reference to a specific embodiment of the present invention. The following steps are as follows: Suspension pellets obtained by centrifuging the culture broth of lactic acid bacteria are suspended in 20 mL of distilled water, and then lysozyme (mg / mL) Lt; RTI ID = 0.0 > 37 C < / RTI > Then, the suspension is disrupted with an ultrasonic pulverizer to obtain a lactic acid bacteria enzyme solution. The lactic acid bacteria enzyme solution is mixed with sodium alginate, and the enzyme is immobilized by dropping 1 drop in 2% calcium chloride solution. The immobilized lactic acid bacteria enzyme solution is centrifuged to remove supernatant, washed with a washing solution (for example, phosphate solution), and stored in a refrigerator.

According to the present invention, when galactooligosaccharide is produced using the immobilized lactic acid bacteria enzyme solution in the whey culture, galactooligosaccharide can be obtained at a yield of 15.4% at the maximum.

According to another aspect of the present invention, there is provided a galactooligosaccharide prepared by the above-described method of the present invention.

In the present invention, there is provided a lactic acid bacterium beverage composition containing galactooligosaccharide produced by the above production method.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a novel method for producing galactooligosaccharides using whey culture medium.

(b) According to the present invention, it is possible to provide galactooligosaccharides which can be ingested concurrently with probiotics.

(c) According to the present invention, there is an advantage that the production cost of galactooligosaccharide can be remarkably reduced compared with the conventional production process.

FIG. 1 is a diagram schematically showing a method for producing a whey powder medium. FIG.
Fig. 2 is a graph showing the amount of liberated o- nitrophenol as a standard curve.
FIG. 3 is a graph showing a standard curve of protein quantitation using bovine serum albumin as a protein standard solution.
4 is a diagram schematically showing a method of immobilizing lactase.
FIG. 5 is a graph showing the absorbance of L. paracasei FBT314 cultured in whey powder medium (RWP) or lactic acid bacteria medium (MRS).
◆: 10% RWP, ●: 15% RWP, □: MRS (control)
FIG. 6 is a graph showing the results of counting colonies after culturing L. paracasei FBT314 cultured in whey powdery medium or lactic acid bacteria medium on a MRS solid medium.
◆: 10% RWP, ●: 15% RWP, □: MRS (control)
7 is a graph showing changes in the growth of L. paracasei FBT314 according to the nitrogen source contained in the whey powder medium.
□: Yeast extract, *: Soiton, ◇: Beef extract, ○: Malt extract
8 is a graph showing changes in the growth of L. paracasei FBT314 according to the concentration of yeast extract contained in the whey powder medium.
?: 0%,?: 0.5%, 1%, X: 1.5%,?: 2%
9 is a graph showing the activity of lactic acid bacteria (LAB) lactase in precipitated pellets or supernatants of lactic acid bacteria cultured in powdered whey culture.
10 is a graph showing changes in the production of galactooligosaccharide according to the concentration of whey powder in the production of galactooligosaccharides using lactase derived from L. paracasei FBT314.
11 is a graph showing changes in the production of galacto-oligosaccharides according to temperature in the production of galactooligosaccharides using lactase derived from L. paracasei FBT314.
12 is a graph showing changes in the production of galactooligosaccharide according to the reaction time in the production of galactooligosaccharides using lactase derived from L. paracasei FBT314.
FIG. 13 is a photograph showing the presence or absence of galactooligosaccharide by thin layer chromatography (TLC) in a sample cultured by adding lactase derived from L. paracasei FBT314 to powdery whey culture medium.
1. Glucose, 2. Galactose, 3. Lactose, 4. Galactooligosaccharide
5. Sample (1/2), 6. Sample (1/4), 7. Sample (1/8), 8. Sample (1/16)
14 is a graph showing the results of performing HPLC for quantitative analysis of galactooligosaccharide.
15 is a graph showing the results of quantitative analysis of galactooligosaccharide produced by an immobilized enzyme using HPLC.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Throughout this specification, "%" used to denote the concentration of a particular substance is intended to include solids / solids (wt / wt), solid / liquid (wt / The liquid / liquid is (vol / vol)%.

Experimental material

Materials and badges

The whey powder used in the present invention was obtained in the manufacture of cheddar cheese for processed cheese in Korea and supplied by Samky Oil Co., Ltd. The whey medium was used in two different conditions: a medium for lactic acid bacteria culture and a medium for the production of galactooligosaccharide. Yeast extract (yeast extract, BD, USA) and ammonium citrate (Shinyo pure chemicals, Japan) were used as additives for lactic acid bacteria culture medium. The lactose was adjusted to 1.75%. The manufacturing method is shown in FIG. 1 and Table 3. The composition of the whey medium for the production of galactooligosaccharide was sterilized at 90 ° C for 20 minutes by adjusting the lactose to 30% and the composition was as shown in Table 4. The MRS medium was purchased from BD Company (USA), and the standard reagent was purchased from Sigma (USA).

Composition of medium for growth of lactic acid bacteria Powder whey 25 g Alcalade 0.1 g Yeast extract 10 g Citric acid ammonium salt 2 g Distilled water (total) 1 L

Powdered Whey: Samik Dairy company (Denmark), Alcalade: Novozyme (Denmark)

Composition of reaction solution for the production of galactooligosaccharide Powder whey 430 g Sodium azide (NaN ₃ ) 0.5 g Distilled water (total) 1 L

Disclosed strains and enzymes

The microorganisms having lactase activity were able to utilize lactose and also had a function of synthesizing galacto-oligosaccharides. Therefore, lactose-utilizing lactic acid bacteria capable of proliferating lactose as a sole carbon source were firstly selected. As the selected lactic acid bacteria, Lactobacillus strain isolated from commercially available yogurt paracasei FBT314, Lactobacillus helveticus FBT1 and Lactobacillus acidophilus KCCM32820, which are the strains of the present inventors, Lactococcus lactis ATCC11454, Lactobacillus rhamnosus KCCM32405 , Lactobacillus brevis KCCM11904 , Lactobacillus confusus KCCM40015 , Lactobacillus reuteri ATCC23272 , Lactobacillus delbrueckii KCCM35468 , Lactobacillus plantarum KCCM12116 , Leuconostoc mesenteriodes KCCM 11324 and Weissella kimchii KCCM41287 and 12 weeks old lactic acid bacteria. 12 weeks. Lactic acid bacteria were cultured for two times in MRS (BD, USA) modified medium, and then used for 1 time using lactic acid bacteria culture whey culture medium.

For lactic acid bacteria Whey medium  In manufacturing Whey powder  Effect of Concentration

A liquid medium containing 10% and 15% by weight of whey powder was prepared, and a control medium was MRS (BD, USA) liquid medium. Lactobacillus was inoculated and incubated at 37 ° C for 60 hours. The growth of the bacteria was measured at 650 nm using a spectrophotometer (Thermo Scientific, USA).

For lactic acid bacteria In whey medium  Proliferation of lactic acid bacteria and Viable cell count  Measure

Reduced Whey Powder Medium (RWP) and MRS Liquid Medium were prepared and inoculated with lactic acid bacteria, cultured at 37 ° C for 60 hours, plated on MRS solid medium, incubated at 37 ° C for 60 hours and grown on solid medium The number of typical colonies was counted.

For lactic acid bacteria Whey medium  Effect of organic matter on manufacturing

(Yeast extract, BD, USA), soytone (BD, USA), beef extract (BD, USA) were used to investigate the growth of microorganisms according to various nitrogen source. , And malt extract (BD, USA) were added to the culture medium, and the culture broth was inoculated with the culture medium. Then, the growth of the cells was indicated by absorbance at 650 nm for 60 hours.

For lactic acid bacteria Whey medium  Effect of Yeast Extract on Manufacturing

Absorbance was measured at 650 nm using a spectrophotometer with the amount of yeast extract added at 0, 0.5, 1, 1.5, and 2% as the organic wastes lacking in whey powder.

Identification of lactase enzyme activity of lactic acid bacteria

Twelve kinds of lactic acid bacteria were cultured in MRS medium and then 1 mL of each was taken and centrifuged (2000 × g, 20 minutes) with a centrifuge (Vision, Korea). The supernatant was taken separately and resuspended in 1 mL of saline (0.85% NaCl) in pellet. After centrifugation, 1 mL of saline was added to the precipitated cells and suspended. Add 0.1 mL of ONPG disc (Sigma, USA) and 0.1 mL of saline to the sterilized test tube, add 0.1 mL of the supernatant and cell suspension prepared as described above, shake thoroughly, and incubate at 30 ° C. The enzyme activity was measured by measuring the amount of o-nitrophenol (Sigma, USA) liberated from o-Nitrophenyl-β-D-galactopyranoside (ONPG, Sigma) by modifying the method of Nguyen et al. 0.5 mL of enzyme solution and 100 mM phosphate buffer solution (pH 7.0) were mixed in a 1.4 mL empty test tube and mixed with 0.1 mL of 20 mM ONPG solution. The mixture was reacted for 10 minutes in a constant temperature water bath at 30 ° C. After 10 minutes, 0.2 mL of 0.2 M Na ₂ CO ₃ solution was added to stop the reaction, and the absorbance at 405 nm was measured. Enzyme 1 U was defined as the number of micromoles of o-nitrophenol produced from ONPG per reaction time. The amount of o-nitrophenol liberated from the enzyme unit was calculated from a standard curve prepared in advance (Fig. 2).

Lactic acid bacteria  Used Crude enzyme solution  Produce

Lactic acid bacteria were inoculated in 10 mL of MRS liquid medium and incubated at 30 ° C. for 18 hours. The cells were inoculated into 100 mL of liquid powdery whey culture medium and cultured at 30 ° C. for 18 hours. The culture broth of lactic acid bacteria was centrifuged at 2000 × g for 20 minutes, the supernatant was discarded, suspended in pellet in distilled water, and centrifuged under the same conditions. The supernatant was discarded, 20 mL of distilled water was added to the pellet, 1 mg / mL of lysozyme was added, and the mixture was incubated at 37 ° C for 1 hour. The cells were disrupted using an ultrasonic disintegrator (Sonics, USA). Add 1.4 μL of 100 mM phosphate buffer (pH 7.0) aseptically to the aseptic solution, add 100 μL of the crude enzyme solution and 100 μL of 20 mM ONPG stock solution, incubate at 30 ° C for 10 minutes, add 0.2 M Na ₂ CO ₃ After 200 μL of the reaction was stopped, the absorbance was measured at 405 nm using a spectrophotometer.

Effect of temperature on lactase activity

to a 100 mM phosphate buffer, pH 7.0 mixed and the resulting lactic acid crude enzyme solution and the 20, 25, 30, 35 and 10 minutes of reaction the ONPG in 40 ℃ condition, to stop a 0.2 M Na ₂ CO ₃ solution, the reaction was added to 0.2 mL Absorbance was measured at 405 nm.

For lactase activity pH Influence of

The pH of the 100 mM phosphate buffer was adjusted to 6.0, 6.5, 7.0, and 7.5, and the mixture was mixed with lactic acid bacteria-derived crude enzyme solution, and ONPG was reacted for 10 minutes. 0.2 mL of 0.2 M Na ₂ CO ₃ solution was added to stop the reaction, Absorbance was measured.

Crude enzyme solution  Protein quantification

The concentration of crude enzyme derived from lactic acid bacteria was measured using the Bio-Rad protein assay kit (Bio-Rad) by Bradford method (1976). Bovine serum albumin (Bio-Rad) was used as a protein standard solution, and the absorbance was measured at 595 nm using a spectrophotometer (FIG. 3).

TLC Using Galactooligosaccharide  Confirm

In order to determine the optimum temperature for the production of galactooligosaccharides from lactase derived from L. paracasei FBT314, which was finally screened, we modified the method of Itoh et al. (1982). The whey powder for the production of galactooligosaccharide was prepared in 15 mL of each of 10, 20 and 30% whey powder, and 1, 3 and 5 mL of crude enzyme solution (206 U / protein mg) For 2, 4, 6, and 8 hours to determine the presence or absence of galactooligosaccharide production on TLC (Merck, Germany). The developing solvent was isopropanol-water (4: 1). As the coloring reagent, 5 mL of a solution of 1% of aniline dissolved in acetone and 1% of diphenylamine in acetone were mixed and 85% of H ₃ PO _{4 was added} to 1 mL.

Of galactooligosaccharide  Identification and Quantification

To quantify the amount of galactooligosaccharide, 15 mL of sample was boiled with a certain amount of activated carbon, filtered once using filter paper (Whatman), transferred to a syringe and filtered through a 0.45 μm syringe filter (Advantec, Japan) Chromatography (HPLC, Waters, USA) was used and the analytical conditions are shown in Table 5.

Operating Conditions of HPLC for Galactooligosaccharide Analysis Name of device Waters e2695 Detector RI detector column YMC-Pack Polyamine II Oven temperature 25 ℃ Mobile phase Acetonitrile: Water (75: 25, v / v%) Flow rate 1.0 mL / min Injection volume 20 μL

Lactase Crude enzyme solution  Immobilization

Sodium alginate (Yakuri, Japan) was used for the experiment to confirm the optimum condition of the immobilization support by modifying the method of Jang et al. (1990) (Table 6). The optimum combination was determined by measuring the viscosity of the coating material prepared at the concentration of 1, 1.5, and 2% of alginate soda. The enzyme was immobilized by dropping the crude enzyme solution into the 2% CaCl ₂ solution dropwise using a syringe. The immobilization method is shown in Fig. The prepared beads were washed several times with 100 mM phosphate buffer (pH 7.0) and stored at 4 ° C for the experiment.

Coating materials for lactase immobilization number Coating material One 1% sodium alginate 2 1.5% sodium alginate 3 2% sodium alginate

Experiment result

Whey medium  The concentration of lactose and the growth of lactic acid bacteria

To determine the growth of lactic acid bacteria according to the lactose content of the whey medium, it is generally known that the whey powder contains 70% of the lactose content. The results of comparative measurement of the absorbance of lactic acid bacteria inoculated with lactic acid bacteria in MRS medium prepared by adding whey powder at 10 and 15% and culturing for 60 hours are shown in FIG. In addition, a certain amount of bacteria was taken at each time of measuring the absorbance, and the colonies were counted by culturing on MRS solid medium and culturing at 30 DEG C for 60 hours. The results are shown in FIG. The lactose content of the whey cultured lactic acid bacteria was similar to that of the lactic acid bacteria MRS and 10% whey powder, and the culture medium of 15% whey powder was found to be lower than the other two cultures. This is probably because lactase activity in the lactic acid bacterium increases the activity of binding galactose to other lactose rather than the activity of degrading lactose into glucose and galactose when the concentration of lactose is increased.

Effect of Organic Nitrogen Source on the Growth of Lactic Acid Bacteria

To determine the growth of lactic acid bacteria according to the type of nitrogen source in the whey culture, 1% of yeast extract, soybean, beef extract and malt extract were added as a nitrogen source to the lactic acid bacteria as a basic medium and cultured for 60 hours And the absorbance was measured by taking the sample in 12 hour unit. As shown in Fig. 7, there was almost no difference in the growth of the lactic acid bacteria depending on the four nitrogen sources. The growth of lactic acid bacteria was measured for up to 60 hours with 0%, 0.5%, 1%, 1.5% and 2% of yeast extracts as the most preferred yeast extract as the organic waste lacking in the whey powder. , And it was found from FIG. 8 that the growth of lactic acid bacteria was superior in the sample to which the nitrogen source was added as compared with the sample to which no nitrogen source was added. The growth of almost the same bacteria in all samples up to 6 hours showed that the samples without added nitrogen source grew bacteria through the proteins present in the powdery whey and after 6 hours the consumption of nitrogen source in the powdery whey And 1% of nitrogen source was considered to be the best when considering the growth of lactic acid bacteria by price. In the subsequent experiment, 1% yeast extract was added as a nitrogen source, and whey culture medium for lactic acid bacteria culture was added .

Determination of Lactic Acid Bacterium Lactase for Selection of Optimal Lactic Acid Bacteria

In order to examine the lactase activity of the 12 weeks-old lactic acid bacteria isolated by the present inventors, the results were confirmed by ONPG discs under the conditions of the precipitated cells and the supernatant, as shown in Table 7-8 and FIG. The enzyme activity of the lactase was confirmed in the precipitated cells, but the lactase activity was not confirmed in the supernatant. This suggests that the lactacase derived from lactic acid bacteria is present in the cells, not in the cells. According to Nguyen et al. (2007), L. acidophilus lactase was considered to be present in cells, and cells were disrupted using a French press (Aminco, Maryland). In addition, enzyme activity was confirmed in most of the 12 kinds of lactic acid bacteria used in the experiment, but the activity of Leuconostoc mesenteriodes KCCM 11324 and Weissella kimchii Lactase enzyme activity was not confirmed by KCCM41287. This is because lactic acid bacteria present in kimchi are mostly lactic acid bacteria present in plants, and therefore, they do not have the function of degrading lactose or producing galactooligosaccharides from lactose.

Identification of LAB Lactase Activity in Precipitated Cells number Lactobacillus Experiment result One Lactobacillus rhamnosus KCCM32405 + 2 Lactobacillus brevis KCCM11904 + 3 Lactobacillus confusus KCCM40015 + 4 Lactobacillus reuteri ATCC23272 + 5 Lactobacillus delbrueckii KCCM35468 + 6 Lactobacillus acidophilus KCCM32820 + 7 Lactobacillus plantarum KCCM12116 + 8 Lactococcus lactis ATCC11454 - 9 Leuconostoc mesenteriodes KCCM11324 - 10 Weissella kimchii KCCM41287 - 11 Lactobacillus helveticus FBT1 + 12 Lactobacillus paracasei FBT 314 +

+, positive; -, negative

Identification of activity of LAB lactase in culture supernatant number Lactobacillus result One Lactobacillus rhamnosus KCCM32405 - 2 Lactobacillus brevis KCCM11904 - 3 Lactobacillus confusus KCCM40015 - 4 Lactobacillus reuteri ATCC23272 - 5 Lactobacillus delbrueckii KCCM35468 - 6 Lactobacillus acidophilus KCCM32820 - 7 Lactobacillus plantarum KCCM12116 - 8 Lactococcus lactis ATCC11454 - 9 Leuconostoc mesenteriodes KCCM11324 - 10 Weissella kimchii KCCM41287 - 11 Lactobacillus helveticus FBT1 - 12 Lactobacillus paracasei FBT 314 -

+, positive; -, negative

GOS  Lactic Acid Lactase Selection for Production

Based on the results of lactase enzyme activity measurement using ONPG, four probiotic lactic acid bacteria, which are widely used as starters of commercially produced yogurt in Korea, were selected and tested. Table 9 shows the results of measuring lactase enzyme activity of the four kinds of the disclosed lactic acid bacteria. The enzyme activity of lactic acid bacteria was measured at about 102-1,053 units, and the enzyme activity of lactase derived from L. paracasei FBT314 was the highest. The protein content of this lactase enzyme solution was measured to be 5.1 mg / mL, and the specific activity of lactase derived from L. paracasei FBT314 was calculated as 206 U / protein, mg. Nguyen et al. (2006, 2007) according to L. acidophilus and L. reuteri in the lactase activity of the resulting 230, 180 L. paracasei used in the present invention as U / mg for There was no significant difference from lactacase derived.

Comparison of Lactase Activity of Lactic Acid Bacteria LAB system L. acidophilus Lc . lactis L. helveticus L. paracasei Enzyme activity (U) ^* 228 102 520 1,053

^* Enzyme 1 U was defined as the number of micromoles of o-nitrophenol produced from ONPG per reaction time.

L. paracasei FBT 314  For lactase activity pH Influence of

L. paracasei The buffers were prepared at pH 6.0, 6.5, 7.0 and 7.5 in order to investigate the effect of pH on the lactase activity produced by FBT314. The ONPG test was carried out in the same manner as in Table 10. As a result, L. paracasei The activity of lactase from FBT314 was the highest at pH 6.5-7.0. The activity of lactase produced by L. paracasei FBT314 was high at neutral pH. Hung et al. (1994) reported that Bifidobacterium The optimal pH of infantis- derived lactase was reported to be 6.5. Hinz et al. (2004) had reported the pH optimum of the lactase derived from B. adolescentis to 6.0, Nguyen et al. (2007) optimum pH of lactase derived from L. acidophilus was reported as 6.5 to 8.0.

Effect of pH on the activity of L. paracasei FBT314 lactase pH 6.0 6.5 7.0 7.5 Enzyme activity (U) ^* 104 1191 1273 363

^* Enzyme 1 U was defined as the number of micromoles of o-nitrophenol produced from ONPG per reaction time.

L. paracasei FBT 314  Effect of temperature on lactase activity

L. paracasei The ONPG test was carried out at 20, 25, 30, 35 and 40 ℃ in order to investigate the influence of temperature on the lactase activity produced by FBT314. L. paracasei The optimum temperature of the lactase derived from FBT314 was 30 占 폚. This is because L. paracasei Since FBT 314 is mesophilic bacteria, it was judged that the lactic acid-derived lactase also had a high enzyme activity at about 30 ° C.

Influence of Temperature on the Activity of L. paracasei FBT314 Lactase Temperature (℃) 20 25 30 35 40 Enzyme activity (U) ^* 216 465 1254 1162 1068

^* Enzyme 1 U was defined as the number of micromoles of o-nitrophenol produced from ONPG per reaction time.

Galactooligosaccharide  In production Whey powder  Effect of Concentration

To determine the conditions under which the lactase produced the lacto-oligosaccharide from lactose, the whey powder concentration was adjusted to 20, 25, 30, 35 and 40% to prepare the powdered whey culture medium for the production of galacto-oligosaccharide, and L. paracasei The results of quantitative measurement of the production of galactooligosaccharide by adding FBT314-derived lactase were as shown in Fig. The production of galactooligosaccharide increased sharply to 30% and gradually increased after 30%. As a result, it was concluded that the optimum concentration of the whey powder was 30%.

Galactooligosaccharide  Influence of temperature on production

The results of experiments at 20, 30, 40, 50, and 60 ° C to determine the optimum galactooligosaccharide production temperature of L. paracasei FBT314-derived lactase are shown in FIG. It was found that the production of galactooligosaccharide gradually decreased after 30 ° C. As in the case of the lactase enzyme activity measurement by the ONPG test, 30 ° C. showed L. paracasei It was found that the optimum temperature of FBT314-derived lactase was found.

Galactooligosaccharide  Effect of reaction time on production

In order to confirm the yield of galactooligosaccharide production over time, the results of measurement up to 0, 2, 4, 6 and 8 hours are shown in FIG. As the time increased, the yield of galactooligosaccharide production was also increased. Lactic acid derived from L. paracasei FBT314 is preferably cultured for 4 hours or more in order to produce galactooligosaccharides from powdered whey medium, but the increase is not large after 4 hours. In this way, the optimal condition is 4 hours in terms of economy.

L. paracasei FBT 314  Using lactase Of galactooligosaccharide  Production and quantification

30% powder after producing the whey medium was used as a culture product for 4 hours at the addition of the L. paracasei FBT314 originated lactase and 30 ℃ the sample. The presence or absence of the galacto-oligosaccharide of the sample was confirmed by TLC (FIG. 13). Glucose, galactose, lactose and commercial galacto-oligosaccharides were used as control. In Fig. 13, 5-8 is a result of diluting the sample and spotting it. Glucose, galactose and lactose spots are present in 7-8 and several spots under lactose spots are present, which is the presumption that they are galactooligosaccharides. Since galactooligosaccharide is not a single substance but encompasses 3 mer-5 mer, it is judged that there are several such spots. HPLC was used to quantitatively analyze the resulting galactooligosaccharide (Fig. 14). 70% of the 30% powdery whey medium is lactose, which is primarily degraded by lactase to become glucose and galactose, and galactose is converted to lactose by sugar transfer which binds to other lactose molecules that are not degraded by lactase Lacto oligosaccharide is produced. Therefore, in Fig. 14, the amount of glucose was found to be larger than that of the former galactose. Also, the peak of galactooligosaccharide was confirmed. Lactic acid from L. paracasei FBT314 was treated with 30% powdered whey medium to produce galactooligosaccharides up to 19.41% (Table 12).

Composition of carbohydrates in the production of galactooligosaccharides using lactase number Fructose Glucose Galactooligosaccharide-disaccharide Lactose Galactooligosaccharide-trisaccharide 4'-galactosyllactose 6'-galactosyllactose Galacto-oligosaccharides-sugar chains < Total galactooligosaccharide One 0.76 5.13 1.66 80.73 1.70 3.59 1.24 4.65 12.84 2 0.47 2.41 1.42 86.7 0 4.13 0.47 4.01 10.03 3 0.51 6.21 1.74 81.58 0.23 4.10 1.37 3.94 11.38 4 0.56 6.60 2.07 80.07 0 3.95 1.84 4.61 12.47 5 0 5.38 1.56 82.33 1.80 3.65 1.10 4.18 12.29 6 0 4.06 5.04 79.81 2.20 4.05 1.14 3.70 16.13 7 0 5.25 6.44 75.34 0 5.02 1.91 6.04 19.41 8 0 2.34 5.50 83.69 0 4.39 0.58 3.50 16.31 9 0.53 9.06 2.09 73.80 2.65 4.26 2.48 4.82 16.3 10 0.5 7.60 1.03 78.69 2.5 4.12 0.76 4.62 13.03 11 0.32 7.81 1.19 79.61 1.78 3.51 0.75 4.83 12.06 12 2.12 8.95 0.73 76.95 1.8 3.39 0.74 5.14 11.8

Total galacto-oligosaccharides = galacto-oligosaccharides-disaccharides + galactooligosaccharides-trisaccharides + 4'-galactosyllactose + 6'-galactosyllactose + galactooligosaccharides-

Viscosity measurement of coating material for immobilization of enzyme

The viscosity of the coating material prepared by mixing 1, 1.5 and 2% sodium alginate (Yakuri, Japan) with 1, 3 and 5% modified starch in 1% sodium alginate was measured and the results are shown in Table 13 . As the concentration of alginate soda increased, the viscosity increased. The enzyme activity of the immobilized enzyme was measured and the results are shown in Table 14. When the viscosity of the immobilized material was low, the immobilization was not performed well, and when the viscosity of the immobilized material was high, the activity of the enzyme was low. When the lactase derived from L. paracasei FBT314 was fixed with sodium alginate, the concentration of 1.5% was considered to be most appropriate.

Alginate Soda (%) 1.0 1.5 2.0 Viscosity (cP) 25.0 ± 0.3 38.4 ± 0.2 61.2 ± 0.5

Alginate Soda (%) 1.0 1.5 2.0 Enzyme activity (U) 583 ± 2 1,147 ± 5 361 ± 1

Immobilized enzyme-produced Of galactooligosaccharide HPLC  Quantitative analysis

15 and 15 are results of quantitative analysis of galactooligosaccharide produced by immobilized enzyme using HPLC. 15 shows the peak of galactooligosaccharide. In Table 15, it was confirmed that the yield of galactooligosaccharide was about 15.4% when the galactooligosaccharide was produced using an enzyme immobilized with the crude enzyme from L. paracasei FBT314. There was a large difference in peak yield from the crude enzyme solution which was not immobilized, but the difference was not significant on average.

The composition of carbohydrates in the production of galactooligosaccharides using immobilized lactase number Fructose Glucose Galactooligosaccharide-disaccharide Lactose Galactooligosaccharide-trisaccharide 4'-galactosyllactose 6'-galactosyllactose Galacto-oligosaccharides-sugar chains < Total galactooligosaccharide One 1.06 21.55 1.92 61.77 2.38 4.39 2.08 4.64 15.41 2 2.22 10.62 1.41 71.67 1.88 4.52 1.45 5.37 14.63 3 1.99 16.32 1.35 67.88 1.9 3.78 1.42 5.03 13.48 4 4.82 18.79 2.21 65.96 0 4.58 1.85 1.80 10.44 5 0.90 19.62 1.77 65.95 2.45 4.03 1.57 4.38 14.2 6 2.1 20.15 1.27 62.02 2.18 4.03 1.57 6.34 15.39 7 0.32 14.24 0.18 70.60 2.8 3.79 1.69 6.1 14.56

Total galactooligosaccharide-galactooligosaccharide-2 saccharides + galactooligosaccharide-3 saccharides + 4'-galactosyllactose + 6'-galactosyllactose + galactooligosaccharide-4 saccharides <

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (11)

(a) culturing a lactic acid bacterium in a whey culture medium for culturing a lactic acid bacterium comprising 10 to 15% by weight of a whey powder and 0.5 to 2% by weight of an organic matter; And
(b) applying the lactic acid bacterium to a whey culture medium for producing galactooligosaccharide containing 20 to 40% by weight of whey powder to produce galactooligosaccharide,
Wherein the production of the galactooligosaccharide is carried out at a pH of 6.5 to 7.0 and at a temperature of 25 to 35 ° C.
The method according to claim 1,
A method for producing a galactooligosaccharide, which further comprises the step of producing a lactic acid bacterial enzyme solution from the lactic acid bacteria between steps (a) and (b).
The method according to claim 1,
Wherein the organic matter contained in the whey culture medium for lactic acid bacteria culture is a yeast extract.
The method according to claim 1,
The lactic acid bacterium may be selected from the group consisting of Lactobacillus paracasei ), Lactobacillus ( Lactobacillus rhamnosus ), Lactobacillus brevis , Lactobacillus &lt; RTI ID = 0.0 &gt; confusus , Lactobacillus reuteri , Lactobacillus &lt; RTI ID = 0.0 &gt; delbrueckii , Lactobacillus &lt; RTI ID = 0.0 &gt; acidophilus , Lactobacillus &lt; RTI ID = 0.0 &gt; plantarum ), Lactococcus lactis lactis , Lactobacillus &lt; RTI ID = 0.0 &gt; helveticus and Leuconostoc mesenteriodes. &lt; RTI ID = 0.0 &gt; 21. &lt; / RTI &gt;
The method according to claim 1,
Wherein the lactic acid bacterium is Lactobacillus paracasei .
3. The method of claim 2,
Wherein the lactic acid bacteria enzyme solution comprises lactase.
3. The method of claim 2,
Wherein the lactic acid bacteria enzyme solution is immobilized.
8. The method of claim 7,
Characterized in that sodium alginate is used as the immobilized enzyme for immobilization.
9. The method of claim 8,
Wherein the alginate soda is used in an amount of 1 to 2% by weight.
A galactooligosaccharide produced by the method of any one of claims 1 to 9.
A lactic acid bacterium beverage composition containing the galactooligosaccharide of claim 10.

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