KR20140012932A - Method for preparing galactooligosaccharide using lactobacillus extract and whey - Google Patents

Abstract

The present invention relates to a new production method of galacto-oligosaccharides, which includes a step for culturing lactobacillus in a whey medium for culturing Lactobacillus, and a step for producing galacto-oligosaccharides by applying the Lactobacillus to a whey medium for producing the galacto-oligosaccharides. According to the present invention, a user can provide galacto-oligosaccharides which can be eaten with probiotics at the same time, and the production costs of the galacto-oligosaccharide can be remarkably reduced compare to exiting production processes.

Description

Method for preparing galactooligosaccharides using lactic acid bacteria extract and whey {Method for Preparing Galactooligosaccharide Using Lactobacillus Extract and Whey}

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

Lactose, the major carbohydrate in milk and dairy products, is a disaccharide consisting of glucose and galactose, which is contained only in the milk of all mammals, and contains 4-5 g of lactose per 100 g of milk. However, lactose is a nutritionally problematic substance for the majority of African-American and Asian sulfur adults. The lack of enzymes that can break down lactose in the small intestine prevents the breakdown of ingested lactose, resulting in lactose intolerance or lactose intolerance that causes symptoms such as bloating, abdominal pain, cramps and diarrhea. It has led to problems such as reduced consumption and poor quality 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 curds during cheese production. As whey has increased in recent years, production has also increased and is a dairy processed by-product that is recognized as an important substitute for skim milk powder. The average amount of whey produced per kilogram of cheese is 9 kilograms, nine times the cheese output (Yoon et. al ., Korean J. Food Sci , Ani . Resour ., 17 (1), 38-44 (1997)).

Lactobacillus is well known as "probiotics" that are present in the small intestine to inhibit the growth of harmful bacteria by lowering the pH of the intestine by secreting lactic acid as the lactic acid bacteria grow. Lactobacillus acidophilus is the first probiotics to be discovered, and there have been active studies on the probiotics efficacy of other lactic acid bacteria. Lactobacillus also reduces lactose intolerance (Mcdonough et. al ., Am . J. Clin . Nutr ., 45, 570-574 (1987)) and its role in lowering cholesterol (Holzapfel et. al ., Int J Food Microbiol . 26; 41 (2): 85-101 (1998)), and the reduction of the risk of colorectal cancer (Sanders, Int . Dairy J. , 8, 341-347 (1998)).

  As the standard of living improves, there is a growing interest in health, and domestic food companies are making great efforts to develop safe natural food materials. As a result of these efforts, various oligosaccharide products have been developed and marketed. Oligosaccharide is a processed sugar solution obtained from the saccharide raw material, currently produced oligosaccharides (fructooligosaccharides), maltooligosaccharides (maltooligosaccharides), isomalttooligosaccharides (galactooligosaccharides, galactooligosaccharides (GOS)) and the like. Fructooligosaccharide refers to a liquid or powder form obtained by filtering, purifying and concentrating a sugar solution obtained by using an enzyme from a sugar solution or a saccharide raw material obtained by acting an enzyme to combine one or more fructose molecules. Refers to a liquid or powder form obtained by filtering, purifying and concentrating a sugar solution obtained by acting an enzyme so that a molecule has a basic structure of branched bond. In addition, galactooligosaccharide refers to a liquid or powder form obtained by filtering, purifying, and concentrating a sugar solution of raffinose and starchose, which is obtained from a transgalgaltooligosaccharide liquid obtained from an action of an enzyme using a saccharide raw material or sugar beet, soybean, and the like. It refers to a liquid or powdered form of a sugar solution obtained by operating an enzyme such that 3-10 glucose molecules are directly linked using a saccharide raw material (Food Code, 2009).

These oligosaccharides do not decompose in the small intestine and go to the large intestine to act as a growth factor for the intestinal useful bacterium Bifidobacteria , thereby inhibiting the growth of intestinal Escherichia coli, enterococci and decaying bacteria. In addition, it is useful for people whose sugar intake is restricted due to diabetes, as it shows the effect of constipation according to the synthesis of nutrients, promoting metabolism, enhancing immune function, and promoting bowel movement. It is also used as food ingredients, pharmaceuticals, and livestock feeds such as formula, milk powder, beverages, ice cream and bread (Kim CR, Korean J. Food). Nutr ., 12 (1), 50-54 (1999)).

Beta-galactosidase (EC 3.2) has long been used for the hydrolysis of lactose to produce galactooligosaccharides, one of the oligosaccharides, as the growth factor of the intestinal beneficial bacteria bifidus bacteria. 1.23) through the transgalactosidation / transgalactosylation reaction (In et) al ., Korean J. Food Sci . Technol ., 29 (2), 376-378 (1997). In particular, galactooligosaccharides are present in large amounts in the components of breast milk and are widely used as additives such as dairy products in the food industry. Beta-galactosidase, an enzyme that produces galactooligosaccharide, is also called lactase and is produced by a wide range 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)). As yeast, Cryptococcus laurentii (Ohtsuka et 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)). Molds 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 and others have been studied (Table 1-2).

Biochemical Characteristics of Microbial Beta-galactosidase Temperature rating Enzyme-derived strain Temperature
(℃) pH activation
(U mg ^-1 ) Reference Basophilia
(Psychrophile) Arthrobacter
psychrolactophilus 10 8.0 20 Nakagawa et al . 2007 Mesophilic bacteria
(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 fragilis 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 galactoligosaccharides from galactose using microbial beta-galactosidase Biocatalyst Enzyme derived Temperature
(℃) pH Lactose
(g L ^-1 ) GOS ^a ,%
(Conc, g L ^-1 ) productivity
(g L ^-1 h ^-1 ) Reference Raw 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 Cells
(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, beta-galactosidase is called lactase and is an enzyme that hydrolyzes the β (1 → 4) bond of lactose into glucose and galactose, and attaches galactose cut from lactose to other lactose. An enzyme that produces galactooligosaccharides (Park et) al ., Appl . Microbiol . Biotechnol ., 85, 1279-1286 (2010)).

In food processing, water-soluble enzymes are generally unstable in their enzyme activity and difficult to separate products and enzymes after the reaction, making it impossible to reuse expensive enzymes. As a solution of this, the immobilization of enzymes is recognized as a very useful method, and research on this is being 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 tried to develop a method for producing galactooligosaccharides at low cost using whey medium. As a result, culturing the lactic acid bacteria in lactic acid bacteria whey culture medium; Applying the lactic acid bacteria to the whey medium for producing galactooligosaccharides has developed a new galactooligosaccharide manufacturing process comprising producing galactooligosaccharides. According to this process, the lactic acid bacteria cultured in whey medium can be applied to the whey medium for producing galactooligosaccharide to produce galactooligosaccharide continuously without further purification process, thereby significantly reducing the production cost compared to the conventional manufacturing process. By confirming, this invention was completed.

Accordingly, it is an object of the present invention to provide a method for producing galactooligosaccharide, which is simpler than a conventional manufacturing process using whey medium and significantly reduces production costs.

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

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention and claims.

According to one aspect of the present invention, the present invention provides a method for preparing galactooligosaccharide using whey medium, comprising the following steps:

(a) culturing the lactic acid bacteria in lactic acid bacteria whey culture medium; And

(b) applying the lactic acid bacteria to the whey medium for producing galactooligosaccharide to produce galactoligosaccharide.

According to one aspect of the invention, the present invention comprises the steps of (a) culturing the lactic acid bacteria in lactic acid bacteria culture whey medium containing 10 to 15% by weight of whey powder and 0.5 to 2% by weight of organic nitrogen source; And (b) applying the lactic acid bacterium to a whey medium for producing galactooligosaccharide comprising 20 to 40% by weight of whey powder to produce galactooligosaccharide.

The present inventors endeavored to develop a method for producing galactooligosaccharide, which significantly reduced the time and production cost compared to the conventional manufacturing process using whey medium. The present inventors cultured the lactic acid bacteria in lactic acid bacteria whey culture medium to obtain lactic acid bacteria, and applied to the whey medium for producing galactooligosaccharide to derive a new galactooligosaccharide production method for producing galactooligosaccharide. The production of lactase required for the production of conventional galactoligosaccharides has been done in mold or yeast, which had to include removing the mold or yeast and purifying the lactase in the final step in order to prevent influx of the fungus or yeast in vivo.

In the present invention, since lactase is produced by lactic acid bacteria, a probiotic beneficial to the living body, it can be used for the production of galactooligosaccharides without a separate purification process. Therefore, a series of processes leading to the cultivation of lactobacillus-containing lactic acid bacteria and the production of galactoligosaccharides can be continuously performed without a separate purification step, thereby saving time and money.

The present inventors culturing the lactic acid bacteria in lactic acid bacteria whey culture medium; And applying the lactobacillus to the whey medium for producing galactooligosaccharide, thereby establishing a new galactooligosaccharide manufacturing process comprising producing galactooligosaccharide, and according to this process, the lactobacillus containing lactase is applied to the whey medium. By producing galactooligosaccharide, a growth factor of beneficial intestinal bacteria, it was confirmed that it was possible to provide galactooligosaccharide capable of simultaneously ingesting beneficial lactic acid bacteria and galactooligosaccharides.

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

Step (a): for lactic acid bacteria culture On whey badge  Cultivation of lactic acid bacteria

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

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

Lactic acid bacteria whey culture for use in the present invention includes a carbon source, nitrogen source and whey components. In the culture medium for lactic acid bacteria of the present invention, various carbohydrates may be used. Preferably, glucose (glucose), sucrose (sucrose), maltose (maltose), fructose (fructose), lactose (lactose), xyllo Ose, galactose or arabinose, more preferably sucrose, fructose, glucose, galactose, arabinose or lactose, most preferably glucose.

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

The whey of the lactic acid bacteria culture whey medium is a liquid remaining after the milk component from the mammal is tangled and solidified, can be used as it is sterilized in a liquid state or powdered by a method such as lyophilization. Whey can be obtained directly from animals or purchased from commercially available products. Such whey may be prepared from, but is not limited to, milk, goat milk, goat oil and camel oil. The whey used in the present invention is preferably powder whey.

According to a preferred embodiment of the present invention, the whey medium for lactic acid bacteria culture is mixed with powdered whey and then added with an alcalase to completely dissolve in a constant temperature bath and sterilized by adding yeast extract and ammonium citrate (ammonium citrate). do.

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

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

Inoculation of lactic acid bacteria is carried out by adding a suitable amount of pre-cultured lactic acid bacteria to the culture medium so that the lactic acid bacteria can fully grow under culture conditions. In the present invention, the medium that can be used for the pre-culture of lactic acid bacteria includes any medium used for the culture of the lactic acid bacteria strain, but preferably, deMan Rogosa Sharpe (MRS) liquid medium, APT (All Purpose with Tween) medium or BHI (Brain Heart Infusion) ) Medium, most preferably MRS liquid medium.

Lactic acid bacteria culture for the production of lactic acid bacteria co-enzyme solution can be carried out by adding 1-5% (v / v) of the pre-cultivated lactic acid bacteria culture medium to lactic acid bacteria culture whey. Cultivation of lactic acid bacteria can be carried out according to the culture conditions of lactic acid bacteria known in the art. This procedure can be easily adapted and used according to strains selected by those skilled in the art (James et al., Biochemical Engineering , Prentice-Hall International Editions). Depending on the growth method of the cells can be suspended or attached culture, depending on the culture method can be carried out in batch, fed-batch or continuous culture.

According to a preferred embodiment of the present invention, after inoculating and culturing the lactic acid strain in the MRS liquid medium, the culture is inoculated in the whey medium for lactic acid bacteria culture. In the present invention, the lactic acid bacteria culture temperature is preferably 20-35 ° C, more preferably 25-32 ° C, and most preferably 28-32 ° C.

With reference to a specific embodiment of the present invention, the step (a) is described as follows: lactic acid bacteria inoculated in 10 mL MRS liquid medium and pre-incubated for 18 hours at 30 ℃, lactic acid bacteria culture medium 100 Inoculate the preculture to mL and incubate for 18 hours at 30 ℃.

Step (b): using lactic acid bacteria Galactooligosaccharide  production

The lactobacillus is applied to the whey medium for producing galactooligosaccharide to produce galactooligosaccharide.

In the present invention, galactooligosaccharide means a generic term for oligosaccharides produced by applying lactase to lactose. For example, the galactoligosaccharide is represented by the general formula Gal- (Gal) n-Glc (Gal means galactose residue, Glc means glucose residue, and n means an integer of 1 to 4). It may be a saccharide to be expressed, Preferably the galactoligosaccharide is galactose to beta-1,4 bonded lactose to lactose 4'-galactosyl lactose, 4'-galactosyl lactose to beta-1,4 bond At least one selected from the group consisting of tetrasaccharides, hexasaccharides, and disaccharides.

The 4'-galactosyl lactose BP gambling compared to raffinose or rakchyuroseu Te Solarium Al Dore sentiseu (Bifidobacterium aldolescentis ), Bifidobacterium bifidum ) or Bifidobacterium breb 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)), improves bioavailability of iron (D. Perez-Conesa et al., Food Science and Technology International , Vol. 13, pp. 69-77 (2007). In addition, Streptococcus , the causative agent of tooth decay It is not used by bacteria such as mutans ) and insoluble glucan is not produced, so it has the advantage of being characterized by low tooth decay (S. Hata. et al., Oral Microbiology and Immunology , Vol 16 pp. 57-62 (2001)), 4'-galactosyl lactose, a representative oligosaccharide of breast milk, is essential for use as a component of infant formula for reproducing metabolic function of breast milk.

Powder whey added to the galactoligosaccharide production whey medium used in the present invention is 20-45% by weight, preferably 25-40% by weight, more preferably 30-40% by weight, most preferably 30-35% by weight.

In the present invention, the step (b) of producing galactooligosaccharide may be performed at a temperature and incubation time range in which the enzyme is not inactivated, and preferably, the step (b) is a culture time and temperature of the enzyme used. It can be performed in a range. Step (b) may be performed for 2-8 hours, more preferably 3-6 hours, most preferably 4-5 hours. In addition, step (b) may be preferably performed at 20-40 ° C., more preferably 25-35 ° C., and most preferably 29-32 ° C.

According to a preferred embodiment of the present invention, between the step (a) and (b) may further comprise the step of obtaining a lactic acid bacteria coenzyme solution containing lactase from the lactic acid bacteria culture solution obtained in step (a). Specifically, centrifugation or filtration may be performed to recover the lactic acid bacteria in the culture and to separate the culture medium, and this step may be performed according to the needs of those skilled in the art. The obtained lactic acid bacteria can be preserved so as not to lose their activity by freezing or freeze drying according to a conventional method. For example, it may be stored at minus 80 ℃ containing glycerol components or suspended in sterile 10% skim milk can be lyophilized. In addition, the microorganism according to the present invention may be improved or improved to have equivalent activity or better activity by conventional physicochemical mutation methods and the like known in the art.

The lactic acid bacteria coenzyme solution used in the present invention is a solution obtained by crushing the lactic acid bacteria obtained by centrifuging the lactic acid bacteria culture medium.

According to the present invention, the lactic acid bacteria coenzyme solution includes lactase. The lactase is an enzyme that hydrolyzes beta-galactosidic bonds and is also called beta-galactosidase.

In the present invention, lactase hydrolyzes galactose and glucose with lactose having a beta-galactoseside as a substrate and simultaneously transfers galactose to lactose to perform galactoligosaccharide production. In addition, lactase produces a galactose enzyme intermediate product containing galactose residues from a lactose substrate, and different products, namely, different kinds of galactoligosaccharides, are generated depending on the substance (galactose receptor) that receives the galactose residues of the galactose enzyme intermediate product. As the reaction proceeds, the reaction product including the galactooligosaccharide generated is further decomposed and recombined to produce various products.

The lactic acid bacteria coenzyme solution used in the present invention may be immobilized to an immobilization carrier. Preferably, the carrier for the correction of the lactic acid bacteria coenzyme solution is 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 sodium alginate used for lactic acid bacterium coenzyme immobilization is preferably 0.5-3.0 wt%, more preferably 1.0-2.0 wt%, and most preferably 1.3-1.6 wt%.

The lactic acid bacteria coenzyme immobilization method of the present invention may use various methods known in the art as a carrier binding method, for example, covalent bonds, ionic bonds, physical adsorption or biochemical bonds.

In the present invention, the step of preparing the lactic acid bacteria coenzyme solution may be carried out within a pH range in which the enzyme is not inactivated, preferably may be carried out in the optimum pH range of the enzyme used. The step can be carried out preferably at pH 6-8, more preferably at pH 6.0-7.5 and most preferably at pH 6.5-7.0.

With reference to a specific embodiment of the present invention, the above steps are described as follows: 20 mL of distilled water is added to the precipitated cells obtained by centrifugation of the lactic acid bacteria culture medium, followed by lysozyme (mg / mL). Treat and leave at 37 ° C. for 1 hour. Then, the suspension is crushed by an ultrasonic grinder to obtain a lactic acid bacteria coenzyme solution. After mixing the lactic acid coenzyme solution with sodium alginate, drop 1 drop into 2% calcium chloride solution to fix the enzyme. The immobilized lactic acid bacteria coenzyme solution is centrifuged to remove the supernatant, washed with a washing solution (eg, phosphate solution) and stored in the refrigerator.

According to the present invention, the production of galactooligosaccharide using lactic acid bacteria coenzyme solution immobilized in whey medium can yield the galactooligosaccharide in a yield of up to 15.4%.

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

In the present invention, a lactic acid bacterium beverage composition containing galactooligosaccharide prepared according to the above method is provided.

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

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

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

(c) According to the present invention, there is an advantage that can significantly reduce the production cost of galactoligosaccharides compared to the conventional manufacturing process.

1 is a diagram schematically showing a method for preparing whey powder medium.
2 is a graph showing the amount of free o- Nitrophenol as a standard curve.
Figure 3 is a graph showing the protein quantification standard curve using bovine serum albumin (bovine serum albumin) as a protein standard solution.
4 is a diagram schematically showing a method of immobilizing lactase.
Figure 5 is a graph showing the absorbance results measured in culture L. paracasei FBT314 in whey powder medium (RWP) or lactic acid bacteria medium (MRS).
◆: 10% RWP, ●: 15% RWP, □: MRS (control)
FIG. 6 is a graph showing colony count after colonizing L. paracasei FBT314 cultured in whey powder medium or lactic acid bacteria medium in MRS solid medium.
◆: 10% RWP, ●: 15% RWP, □: MRS (control)
7 is a graph showing the growth of L. paracasei FBT314 according to the nitrogen source contained in whey powder medium.
□: Yeast extract, *: Soyton, ◇: Beef extract, ○: Malt extract
8 is a graph showing the growth of L. paracasei FBT314 according to the concentration of yeast extract contained in whey powder medium.
▲: 0%, □: 0.5%,: 1%, ×: 1.5%, △: 2%
Figure 9 is a graph showing the activity of lactic acid bacteria (LAB) lactase in the pellet or supernatant of lactic acid bacteria cultured in powder whey medium.
10 is a graph showing the production of galactooligosaccharides according to whey powder concentration in galactooligosaccharide production using L. paracasei FBT314-derived lactase.
FIG. 11 is a graph showing the production of galactooligosaccharides with temperature in galactooligosaccharide production using L. paracasei FBT314-derived lactase. FIG.
12 is a graph showing the production of galactooligosaccharides with reaction time in the production of galactooligosaccharides using L. paracasei FBT314-derived lactase.
FIG. 13 shows the presence or absence of galactoligotang in a sample cultured by adding L. paracasei FBT314-derived lactase to a powder whey medium by thin layer chromatography (TLC).
1. glucose, 2. galactose, 3. lactose, 4. galactoligosaccharide
5. Sample (1/2), 6. Sample (1/4), 7. Sample (1/8), 8. Sample (1/16)
14 is a diagram showing the results of performing HPLC to quantitatively analyze galacto-oligosaccharides.
15 is a diagram showing the results of quantitative analysis of the galactoligosaccharides produced by the 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, unless otherwise indicated, “%” used to indicate the concentration of a particular substance is solid / solid (weight / weight)%, solid / liquid (weight / volume)%, and Liquid / liquid is (volume / volume)%.

Experimental material

Materials and badges

Whey powder used in the present invention is obtained in the domestic manufacturing of cheddar cheese for processed cheese and was provided by Samik Milk Processing Co., Ltd. Whey media were used in two different conditions as lactic acid bacteria culture medium and galactooligosaccharide production medium. The composition of the culture medium for lactic acid bacteria was adjusted to 1.75% of lactose, and as an additive, yeast extract (yeast extract, BD, USA) and ammonium citrate (Shinyo pure chemicals, Japan) were used. 1 and Table 3 and the manufacturing method. The composition of the whey medium for galactooligosaccharide production was adjusted to 30% lactose and sterilized at 90 ° C. for 20 minutes. The composition is shown in Table 4. MRS medium was purchased from BD (USA) and general reagent was purchased from Sigma (USA).

Composition of Medium for Growth of Lactic Acid Bacteria Powder whey 25 g Alcalase 0.1 g Yeast extract 10 g Ammonium Citrate 2 g Distilled Water (Total) 1 L

Powder Whey: Samik Dairy company (Denmark), Alcalase: Novozyme (Denmark)

Composition of Reaction Solution for Galactooligosaccharide Production Powder whey 430 g Sodium azide (NaN ₃ ) 0.5 g Distilled Water (Total) 1 L

Disclosure Strains and Enzymes

The microorganisms with lactase activity were able to use lactose and were also capable of synthesizing galactoligosaccharides. Therefore, lactose-using lactic acid bacteria were selected primarily to magnetize and grow lactose as the only carbon source. Lactobacillus , a strain isolated from commercial yogurt, was selected. paracasei FBT314, Lactobacillus helveticus FBT1 and Lactobacillus acidophilus KCCM32820, Lactococcus lactis ATCC11454, Lactobacillus rhamnosus KCCM32405 , Lactobacillus brevis KCCM11904 , Lactobacillus confusus KCCM40015 , Lactobacillus reuteri ATCC23272 , Lactobacillus delbrueckii KCCM35468 , Lactobacillus plantarum KCCM12116 , Leuconostoc mesenteriodes KCCM11324 and Weissella kimchii The experiment was carried out with a total of 12 strains of Lactic acid strains including KCCM41287. For 12 weeks, Lactobacillus strains were passaged twice in MRS (BD, USA) medium with modified composition, and then passaged once using lactic acid bacteria whey culture.

Lactic acid bacteria Whey badge  In manufacturing Whey powder  Effect of Concentration

Whey powder was prepared by adding 10% and 15% by weight of liquid medium, and the control was MRS (BD, USA) liquid medium. The inoculation of the lactic acid bacteria followed by incubation at 37 ° C. for 60 hours, the growth of the bacteria was measured at 650 nm with a spectrophotometer (Spectrophotometer, Thermo Scientific, USA).

Lactic acid bacteria On whey badge  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, followed by incubation at 37 ° C for 60 hours, taking a certain amount by time, smearing in MRS solid medium and incubating at 37 ° C for 60 hours to grow on solid medium. The number of typical colonies was counted.

* Effects of Organic Nitrogen Sources on the Production of Whey Media for Lactic Acid Bacteria

Yeast extract (BD, USA), soytone (BD, USA), beef extract (beef extract, BD, USA) , 1% of malt extract (BD, USA) was added to prepare a medium, inoculated with lactic acid bacteria, and the proliferation of the cells up to 60 hours was indicated by absorbance at 650 nm.

Lactic acid bacteria Whey badge  Effect of Yeast Extract on Manufacturing

Using the yeast extract as an organic nitrogen source deficient in the powder whey medium, the absorbance was measured at 650 nm using a spectrophotometer with the addition amount of 0, 0.5, 1, 1.5 and 2%.

Confirmation of Lactase Enzyme Activity of Lactic Acid Bacteria

After incubating 12 kinds of lactic acid bacteria in MRS medium 1 mL each was centrifuged (2000 × g, 20 minutes) by centrifuge (Vision, Korea). The supernatant was taken separately and resuspended in 1 mL of saline (0.85% NaCl) in a pellet. After centrifugation, 1 mL of saline was added to the precipitated cells and suspended. Put 0.1 mL of ONPG disc (Sigma, USA) and saline into a sterile test tube, put 0.1 mL of the supernatant and cell suspension prepared in advance as described above, shake well and incubate at 30 ° C. to confirm the color development. Enzyme activity was slightly modified from Nguyen et al. (1999) to determine the amount of o-Nitrophenol (Sigma, USA) liberated from o-Nitrophenyl-β-D-galactopyranoside (ONPG, Sigma). 0.5 mL of enzyme solution and 100 mM phosphate buffer (pH 7.0) were added to a 1.4 mL empty test tube and mixed, and then 0.1 mL of 20 mM ONPG solution was added quickly and mixed, followed by reaction for 10 minutes in a 30 ° C constant temperature water bath. After 10 minutes, 0.2 mL of 0.2 M Na ₂ CO ₃ solution was added to stop the reaction, and the absorbance was measured at 405 nm. Enzyme 1 U was defined as the μmole number of o-Nitrophenol produced from ONPG per minute of reaction time. The enzyme unit was calculated from the standard curve prepared in advance the amount of free o-Nitrophenol (Fig. 2).

Lactobacillus  Used Coenzyme  Produce

The lactic acid bacteria were inoculated in 10 mL of MRS liquid medium and incubated for 18 hours at 30 ° C., and then inoculated in 100 mL of liquid powder whey medium and incubated at 30 ° C. for 18 hours. The lactic acid bacteria culture medium was centrifuged at 2000 × g for 20 minutes, the supernatant was discarded, suspended in pellets with distilled water, and centrifuged again under the same conditions. The supernatant was discarded and 20 mL of distilled water was added to the pellet, lysozyme was added to 1 mg / mL, and then incubated at 37 ° C for 1 hour. The cell solution was disrupted using an ultrasonic mill (Sonics, USA). As a crude enzyme solution, aseptically, 1.4 mL of 100 mM phosphate buffer (pH 7.0) was added, 500 μL of coenzyme solution and 100 μL of 20 mM ONPG stock solution were added, and reacted at 30 ° C. for 10 minutes, followed by 0.2 M Na ₂ CO ₃ After 200 μL of treatment, the enzyme reaction was stopped, and the absorbance was measured at 405 nm using a spectrophotometer.

Effect of temperature on lactase activity

100 mM phosphate buffer of pH 7.0 was mixed with the co-enzyme solution derived from lactic acid bacteria to react ONPG for 10 minutes at 20, 25, 30, 35 and 40 ° C, and 0.2 mL of 0.2 M Na ₂ CO ₃ solution was added to stop the reaction. 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 mixed with the lactic acid bacteria-derived coenzyme solution to react the ONPG for 10 minutes, and 0.2 mL of 0.2 M Na ₂ CO ₃ solution was added to stop the reaction at 405 nm. Absorbance was measured.

Coenzyme  Protein quantification

The coenzyme concentration derived from lactic acid bacteria was measured using a 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 optimal temperature for producing galactooligosaccharides from the lactase derived from the final screened L. paracasei FBT314, it was experimented by modifying the method of Itoh et al. (1982). 15 mL of whey powder for galactooligosaccharide production was prepared using 10, 20 and 30% of whey powder, and crude enzyme solution (206 U / protein mg) was added at 1, 3 and 5 mL, respectively, at 20, 30 and 40 ° C. Incubated for 2, 4, 6 and 8 hours in the presence of galactoligosaccharide production was confirmed by TLC (Merck, Germany). The developing solvent was isopropanol-water (4: 1). As a color developing reagent, 5 mL of 1% of aniline dissolved in acetone and 1% of diphenylamine in acetone were mixed, and 85% H ₃ PO ₄ was added. mL was added and used.

Galactooligosaccharide  Identification and Quantification

To quantify galactooligosaccharides, add a certain amount of activated carbon to 15 mL of sample, boil, filter once using filter paper (Whatman), transfer to syringe, filter with 0.45 μm syringe filter (Advantec, Japan), and put into tube Analysis was performed using chromatography (HPLC, Waters, USA), and the analysis conditions are shown in Table 5.

Operating Conditions of HPLC for Galactooligosaccharide Analysis Device name 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 Coenzyme  Immobilization

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

Coating Materials for Lactase Immobilization number Coating material One 1% Sodium Alginate 2 1.5% Sodium Alginate 3 2% Sodium Alginate

Experiment result

Whey badge  Medium Lactose Concentration and Growth of Lactic Acid Bacteria

In order to determine the growth of lactobacillus according to the lactose content of whey medium, whey powder is known to contain 70% lactose content. 10, 15% of whey powder was prepared to inoculate the lactic acid bacteria in the lactic acid bacteria medium MRS, and cultured for up to 60 hours. In addition, by taking a certain amount of bacteria every time to measure the absorbance and smeared in MRS solid medium and incubated at 30 ℃ for 60 hours to count the colonies are shown in FIG. As a result of controlling the lactose content of the lactobacillus cultured whey medium, the lactobacillus medium showed a similar tendency to the MRS and whey powder 10% medium, and the 15% whey powder medium showed lower growth than the other two mediums. It is thought that this is because, when the concentration of lactose increases, the activity of lactase present in lactic acid bacteria binds to lactose to other lactose rather than to break down lactose into glucose and galactose.

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

In order to examine the growth of lactic acid bacteria according to the type of nitrogen source in whey medium, 1% of yeast extract, soyton, beef extract and malt extract were added as a nitrogen source as a basic medium and inoculated with lactic acid bacteria and incubated for 60 hours. While taking samples in units of 12 hours, absorbance was measured. As shown in FIG. 7, there was almost no difference in the growth of lactic acid bacteria according to four nitrogen sources. It was decided to use the most representative yeast extract as an organic nitrogen source deficient in whey medium, and the growth of lactic acid bacteria was measured up to 60 hours with the amount of yeast extract 0%, 0.5%, 1%, 1.5% and 2%. In Figure 8, it can be seen that the growth of lactic acid bacteria in the sample to which the nitrogen source is added compared to the sample without the nitrogen source. Up to 6 hours, all samples showed almost the same germs. Samples without nitrogen source were grown through protein present in powder whey, and after 6 hours, bacteria were consumed due to consumption of nitrogen source in whey. When the growth of lactic acid bacteria was considered, it was considered that it was best to add about 1% of nitrogen source, and 1% of yeast extract was added as a nitrogen source in future experiments. Prepared.

Confirmation of Lactase in Lactobacillus for the Selection of Optimal Lactobacillus

In order to determine the lactase activity of the lactic acid bacteria of the total 12 weeks isolated by the present inventors, the results confirmed by two conditions of precipitated cells and supernatants through ONPG disc are shown in Tables 7-8 and 9. The enzyme activity of lactase was confirmed in the precipitated cells, but the lactase activity was not confirmed in the supernatant. This suggests that lactase derived from lactic acid bacteria is present in cells rather than excreted outside of cells. Nguyen et al. (2007) determined that L. acidophilus lactase was present in the cells and disrupted the cells using a French press (Aminco, Maryland). You can also check the enzymatic activity of lactic acid bacteria in a week, most of the total 12 kinds of kimchi lactic acid bacteria used in the experiment but a Leuconostoc mesenteriodes KCCM11324 and Weissella kimchii Lactase enzyme activity could not be confirmed in KCCM41287. It was determined that lactic acid bacteria present in kimchi are mostly lactic acid bacteria that existed in plants, so that there is no function of decomposing lactose or producing galactoligosaccharide from lactose.

Activity of LAB Lactase 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 FBT314 +

+, positive; -, negative

Confirmation of LAB Lactase Activity in Cultured 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 FBT314 -

+, positive; -, negative

GOS  Selection of Lactobacillus Lactase for Production

Based on the results of measuring the lactase enzyme activity using ONPG, four probiotic lactic acid strains, which are widely used as starters of commercial yogurt, are currently selected. The results of measuring the lactase enzyme activity of the four test lactic acid bacteria are shown in Table 9. The enzyme activity of lactic acid bacteria was measured at 102-1,053 Units, and the enzyme activity of L. paracasei FBT314-derived lactase was the highest. The protein content of this lactase coenzyme solution was measured to be 5.1 mg / mL, and the inactivation of L. paracasei FBT314-derived lactase was calculated to be 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 the derived lactase.

Comparison of Lactase Enzyme Activity of Disclosed 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 μmole number of o-Nitrophenol produced from ONPG per minute of reaction time.

L. paracasei FBT314  For lactase activity pH Influence of

L. paracasei To determine the effect of pH on lactase activity produced by FBT314, buffers were prepared at pH 6.0, 6.5, 7.0, and 7.5. The results of the ONPG test in the same manner are shown in Table 10. As a result, L. paracasei The enzyme activity of FBT314-derived lactase was the highest at pH 6.5-7.0, indicating that L. paracasei FBT314 produced high enzyme activity at neutral pH. Hung et al. (1994) Bifidobacterium The optimal pH of infantis derived lactase was reported at 6.5. Hinz et al. (2004) reported an optimal pH of B. adolescentis- derived lactase at 6.0, and Nguyen et al. (2007) reported an optimal pH of L. acidophilus- derived lactase at 6.5-8.0.

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

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

L. paracasei FBT314  Effect of temperature on lactase activity

L. paracasei In order to determine the effect of temperature on the lactase activity produced by FBT314, the results of the ONPG test under the conditions of 20, 25, 30, 35 and 40 ℃ are shown in Table 11. L. paracasei The optimum temperature for lactase from FBT314 was found to be 30 ° C. This is L. paracasei Since FBT314 is a mesophilic bacterium, it was determined that the lactic acid bacteria-derived lactase also had high enzymatic activity at about 30 ° C.

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

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

Galactooligosaccharide  In production Whey powder  Effect of Concentration

To determine the conditions under which lactase produces galactoligosaccharides from lactose, whey powder concentrations of 20, 25, 30, 35 and 40% were used to make powdered whey media for galactooligosaccharide production. L. paracasei Quantitatively measuring the production of galactooligosaccharides by the addition of FBT314-derived lactase is shown in FIG. The production of galactooligosaccharides increased sharply to 30% and gradually increased after 30%. Through this, 30% of the whey concentration was determined to be optimal.

Galactooligosaccharide  Effect of temperature on production

In order to confirm the optimum galactooligosaccharide production temperature of L. paracasei FBT314-derived lactase, the results of experiments at 20, 30, 40, 50 and 60 ° C are shown in FIG. 11. Galactooligosaccharide production decreased gradually after 30 ℃, and L. paracasei was found at 30 ℃ as measured by lactase enzyme activity through ONPG test. It was found that the optimal temperature of the FBT314-derived lactase.

Galactooligosaccharide  Influence of reaction time on production

In order to confirm the production yield of galactooligosaccharides over time, the results measured up to 0, 2, 4, 6 and 8 hours are shown in FIG. 12. As time increased, the yield of galactooligosaccharide was also increased. L. paracasei FBT314-derived lactase was recommended to be cultured for more than 4 hours to produce galactooligosaccharides from powder whey media, but the increase was not significant after 4 hours. Through this, the economic condition was 4 hours as the optimum condition.

L. paracasei FBT314  With lactase Galactooligosaccharide  Production and quantification

After preparing 30% whey medium, L. paracasei FBT314-derived lactase was added and the culture product cultured at 30 ° C. for 4 hours was used as a sample. The presence or absence of galactoligosaccharide in the sample was confirmed by TLC (FIG. 13). As a control, glucose, galactose, lactose and commercial galactoligosaccharides were used. 5-8 in Fig. 13 shows the results of spotting the diluted samples. As a result of the development, there are spots of glucose, galactose and lactose in 7-8 and several spots below the lactose spot, which are presumed to be galactoligosaccharides. Since galactooligosaccharides are not one substance but cover 3 mer-5 mer substances, it was judged that there were several spots. HPLC was used to quantitatively analyze the resulting galactoligosaccharides (FIG. 14). 70% of the 30% powdered whey medium is lactose, which is broken down preferentially by lactase into glucose and galactose, of which galactose is broken by sugar metabolism that binds to other lactose molecules not degraded by lactase. Lacto-oligosaccharides are produced. Therefore, in FIG. 14, the amount of glucose was higher than that of the previous galactose. In addition, the peak of galactoligosaccharide was confirmed. Lactose derived from L. paracasei FBT314 was treated in 30% powdered whey media to produce up to 19.41% of galactoligosaccharides (Table 12).

Carbohydrate Composition in the Production of Galactooligosaccharide Using Lactase number Fructose Glucose Galactooligosaccharide-Disaccharide Lactose Galactooligosaccharide-trisaccharide 4'-galactosyllactose 6'-galactosyllactose Galactooligosaccharide-Saccharose < 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 galactoligosaccharide = Galactooligosaccharide-disaccharide + Galactooligosaccharide-trisaccharide + 4'-galactosyl lactose + 6'-galactosyl lactose + galactoligosaccharide-sugars <

Viscosity Measurement of Coating Materials for Enzyme Immobilization

The viscosity of the coating material prepared by mixing 1, 1.5 and 2% sodium alginate (Yakuri, Japan) and 1% sodium alginate in 1, 3 and 5% modified starch is shown in Table 13. . It was found that the viscosity increased as the concentration of sodium alginate increased. The results of measuring the enzyme activity of the immobilized enzyme are shown in Table 14. When the viscosity of the immobilized material was low, the immobilization was not well achieved, and when the viscosity of the immobilized material was high, the activity of the enzyme was found to be low. When the lactase derived from L. paracasei FBT314 was immobilized with sodium alginate, the concentration was determined to be 1.5%.

Sodium alginate (%) 1.0 1.5 2.0 Viscosity (cP) 25.0 ± 0.3 38.4 ± 0.2 61.2 ± 0.5

Sodium alginate (%) 1.0 1.5 2.0 Enzyme Activity (U) 583 ± 2 1,147 ± 5 361 ± 1

Produced with Immobilized Enzyme Galactooligosaccharide HPLC  Quantitative analysis

15 and Table 15 show the results of quantitative analysis of the galactoligosaccharides produced by the immobilized enzyme using HPLC. Referring to FIG. 15, the peak of galactooligosaccharide could be confirmed, and in Table 15, a yield of about 15.4% was obtained when galactooligosaccharide was produced by using an enzyme immobilized with a crude enzyme solution derived from L. paracasei FBT314. There was a big difference in the highest yield from the crude enzyme solution without immobilization, but there was no significant difference on average.

Carbohydrate Composition in Galactooligosaccharide Production Using Immobilized Lactase number Fructose Glucose Galactooligosaccharide-Disaccharide Lactose Galactooligosaccharide-trisaccharide 4'-galactosylactose 6'-galactosylactose Galactooligosaccharide-Saccharose < 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 galactoligosaccharide = Galactooligosaccharide-2 saccharide + Galactooligosaccharide-3 saccharide + 4'-galactosyl lactose + 6'-galactosyl lactose + galactoligosaccharide 4-saccharide <

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 (13)

(a) culturing the lactic acid bacteria in a whey culture medium for lactic acid bacteria comprising 10 to 15 wt% whey powder and 0.5 to 2 wt% organic nitrogen source; And
(B) a method for producing galactooligosaccharide comprising the step of producing the galactooligosaccharide by applying the lactic acid bacteria to the whey medium for galactooligosaccharide production containing 20 to 40% by weight whey powder.
The method of claim 1,
A method for producing galactooligosaccharide further comprising the step of preparing a lactic acid bacteria coenzyme solution from the lactic acid bacteria between steps (a) and (b).
The method of claim 1,
The method for producing galactooligosaccharide, characterized in that the organic nitrogen source contained in the whey culture medium for lactic acid bacteria is a yeast extract.
The method of claim 1,
The lactobacillus is Lactobacillus paracasei paracasei ), Lactobacillus rhamnosus ), Lactobacillus brevis (Lactobacillus brevis), Lactobacillus konpu Versus (Lactobacillus confusus ), Lactobacillus reuteri ), Lactobacillus delbrueckii ), Lactobacillus acidophilus , Lactobacillus platarum plantarum ), Lactococcus lactis ), Lactobacillus helveticus ) and Leuconostoc mesenteriodes ( Leuconostoc mesenteriodes ) is a method for producing galactooligosaccharides, characterized in that selected from the group consisting of.
The method of claim 1,
The lactic acid bacteria is Lactobacillus paracasei ( Lactobacillus paracasei ) method for producing a galactooligosaccharide, characterized in that.
The method of claim 1,
The galactooligosaccharide production method is characterized in that the galactooligosaccharide is carried out at a temperature condition of 25 ℃ to 35 ℃.
The method of claim 1,
The galactooligosaccharide production method according to claim characterized in that carried out at pH 6.5 to 7.0 conditions.
3. The method of claim 2,
The lactic acid bacteria coenzyme solution is a method for producing galactoligosaccharides, characterized in that it comprises a lactase (lactase).
3. The method of claim 2,
The lactic acid bacteria coenzyme solution is a method for producing galactoligosaccharides, characterized in that the immobilization (immobilization).
10. The method of claim 9,
A method for producing galactoligosaccharide, characterized in that sodium alginate is used as an immobilizing enzyme during the immobilization.
11. The method of claim 10,
Sodium alginate is a method for producing galactoligosaccharides, characterized in that used in an amount of 1 to 2% by weight.
Galactooligosaccharide prepared by the method of any one of claims 1 to 11.
The lactic acid bacteria beverage composition containing the galactooligosaccharide of Claim 12.

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