JP2001275561A - Method for producing fermented milk using yolk liquid containing anti-helicobacter pylori antibody - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing fermented milk using the subject yolk liquid maintaining anti-Helicobacter pylori antibody titer and intended for preventing general microorganisms and yeast from contamination. SOLUTION: This method for producing fermented milk comprises the following practice: a saccharide is added to a yolk liquid containing anti-Helicobacter pylori antibody to maintain the antibody titer of the resultant yolk liquid followed by conducting a heat sterilization to prevent general microorganisms and yeast from contamination, and the yolk liquid thus sterilized is mixed into a lactic acid bacteria culture solution followed by agitation.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for producing fermented milk products using an egg yolk solution containing an anti-Helicobacter pylori antibody, while maintaining the antibody titer and preventing contamination of general microorganisms and yeasts. On how you can.

[0002]

2. Description of the Related Art Recently, studies have been actively conducted on injecting an antigen against Helicobacter pylori, which is known as a causative agent of gastric ulcer, into laying hens and using an antibody transferred from this antigen to yolk as an active ingredient. Japanese Unexamined Patent Publication No. 4-
No. 275232 discloses a food for preventing gastric ulcer and duodenal ulcer, which contains an antibody produced using a crushed solution of Helicobacter pylori as an antigen as an active ingredient. Korean Patent Publication 98-8
No. 1323 discloses an antibody yolk antibody immunized against Helicobacter pylori urease and flagella (flagellate) at 0.0
Pharmaceutical for preventing and treating gastritis, gastric ulcer and duodenal ulcer containing 1 to 0.1% by weight and containing an organism selected from lactic acid bacteria, enterococcus, yeast and Bacillus sp. A composition is disclosed. However, when the yolk antibody prepared from the above patents is used for products such as fermented milk and beverages, the heat treatment performed in the process significantly reduces the titer of the specific antibody, and thus the stomach ulcer, gastritis, and 12 It is difficult to expect the effect of prevention and treatment of digital ulcer. As a study for maintaining the titer of the yolk antibody during storage, worms and the like added 30 to 50% by weight of sucrose or sugar to maintain the titer of the antibody during storage or process. C. for 15 minutes (J. of food science, 1).
994, 59 (4), 763-772).

On the other hand, application of chicken eggs to fermented milk is 19
1984 Phosphorus and Cunningham are sterilized and mixed with 47.4% egg white, citric acid, 28.4% alkali-treated soybean oil, and 19.0% skim milk, and then extracted with glucose, sucrose, xanthan gum and vanilla. After adding the ingredients, Lactobacillus bulgaricus and Streptococcus thermophilus were inoculated to develop yogurt. In 1984 Cunningham et al. Sterilized and cooled low-fat homogenous milk,
After homogenizing and mixing ovalbumin sterilized twice at 4 ° C., Lactobacillus bulgaricus and Streptococcus thermophilus were mixed, inoculated, and cultured at 40 ° C. for 6 hours to produce yogurt. In 1983, Kim et al. Added glucose, sucrose, and lactose to a homogeneous whole egg solution under a heat treatment condition of 58 ° C. for 30 minutes, and added lactic acid bacteria to adjust the pH to 4.
The fermented milk was produced by lowering it to as low as 8. In the case of Japan, a patent publication (publication number: 57-54113) discloses that dried and sterilized egg white from which egg white lysozyme has been removed is dissolved in milk, glucose is added, and lactic acid bacteria are inoculated to produce fermented milk. Is disclosed. As a Korean patent, Yu et al. (Publication number: 97-574) is prepared by mixing lactose, sugar, and water with whole egg solution, egg yolk solution, and egg white solution, sterilizing the mixture, and then lactobacillus acidophilus, bifidobacterium longum. ,
A method for producing a lactic acid bacteria fermented beverage that is mixed, inoculated, and fermented with Streptococcus thermophilus has been disclosed. As mentioned above, the development of fermented milk using chicken eggs is whole egg solution, egg white,
To dilute the egg yolk, sugar to promote fermentation, alkali-treated soy milk, skim milk, etc. to produce fermented milk, to resolve the physical stability, such as the layer separation phenomenon of fermented milk,
A stabilizer such as xanthan gum was used.

[0004]

The present invention is different from the conventional technique of adding a lactic acid bacterium growth promoting substance to a diluted solution of chicken eggs and then inoculating lactic acid bacteria and culturing the same.
The present invention relates to a method of adding an egg yolk solution containing an anti-Helicobacter pylori antibody, and more particularly, in the process of producing fermented milk, not only to maintain the anti-Helicobacter pylori antibody titer but also to prevent contamination of general microorganisms and yeast. It is intended to provide a sterilization method.

That is, the present invention is intended to maintain the antibody titer of the yolk solution containing the yolk antibody immunized with the Helicobacter pylori antigen in the production of fermented milk, maintain the antibody titer of the yolk solution, and generally use microorganisms and yeasts. It is an object of the present invention to provide a sterilization method capable of preventing the contamination of water.

[0006]

Means for Solving the Problems The present inventors produced immunized eggs containing antibodies from laying hens using Helicobacter pylori as an antigen. An attempt was made to separate only the yolk portion containing the antibody from the immunized egg thus produced and use it for the production of fermented milk. Then, upon adding the egg yolk liquid to the fermented milk product, to prevent contamination of general microorganisms and yeast, to study sterilization conditions to prevent a decrease in antibody titer during the heat treatment process, to complete the present invention. became.

[0007]

DETAILED DESCRIPTION OF THE INVENTION According to a preferred embodiment of the present invention, a sugar is added to an egg yolk solution containing an anti-Helicobacter pylori antibody to maintain an antibody titer, and sterilization heat treatment is performed to contaminate general microorganisms and yeast. Then, the sterilized egg yolk liquid is mixed with a lactic acid bacteria culture and stirred to produce fermented milk. The preferred sugar used to maintain the antibody titer is selected from the group consisting of sugar, high fructose, and galactooligosaccharides, but is more preferably sugar.

[0008] The content of sugar added to the egg yolk solution is preferably 10 to 30% by weight. In addition, the sterilization heat treatment is performed by 6
Desirably, the reaction is performed 1 to 3 times at 0 to 65 ° C for 2 to 10 minutes.

According to a preferred embodiment of the present invention, the content of the heat-treated egg yolk liquid added to the lactic acid bacteria culture solution is 0.1 to 10% by weight.

Preparation of Antigen Helicobacter pylori, which is known as a causative bacterium of gastric ulcer, was disrupted by ultrasonic waves and centrifuged to obtain a supernatant. The supernatant was ultracentrifuged to precipitate a cell membrane, and then suspended in distilled water and used as an antigen (see Example 1).

Production of Egg Yolk Solution Containing Anti-Helicobacter pylori Antibody A 40-week-old white leghorn was immunized using an outer membrane protein antigen. 9: 1: 8 adjuvant, emulsifier, antigen
After emulsification by mixing at a ratio of 1%, intramuscular injections were performed four times at two-week intervals until sufficient antibodies were obtained. Eggs at the stage where the specific antibody titer transferred to the eggs was sufficiently increased were collected. The collected immunized eggs are separated into albumen and yolk via a cleavage ovum, and the separated yolk is filtered with a filter.
Egg yolk fluid was produced by homogenizing to 80 kg / cm2. The anti-Helicobacter pylori antibody titer of the yolk fluid thus produced was about 5,700 (see Examples 2 and 3).

Antibody titer depending on sugar concentration and sterilization temperature Changes in antibody titer due to sugar concentration and sterilization concentration were confirmed in order to enhance the storage stability of egg yolk fluid and prevent a decrease in antibody titer. As a result, it was shown that the anti-Helicobacter pylori antibody titer can be maintained at 80% or more when the yolk liquid having a sugar concentration of 10% by weight is treated at a sterilization temperature of 63 ° C. or less for 10 minutes (see Example 4). .

Antibody titer and degree of contamination by sterilization time Particularly, the yolk solution to which sugar was added at 10% was added at 63 ° C. for 3, 5,
After sterilization for 7, 10 minutes, the anti-Helicobacter pylori antibody titer and the degree of contamination of general microorganisms and yeast were measured. As a result, when heat treatment was performed at 63 ° C. for 5 minutes, it was shown that the antibody titer was maintained at 90% or more. As a result of the measurement of the degree of contamination, general microorganisms and yeasts were completely removed only by heat treatment for 7 minutes or more. It was confirmed that the cells died (see Example 5).

Antibody Titer and Degree of Contamination According to the Number of Sterilizations The yolk liquid to which 10% of sugar was added was sterilized three times under a heat treatment condition of 63 ° C. for 3 minutes, and the change in the antibody titer and the degree of contamination were confirmed. As a result, it was shown that the anti-Helicobacter pylori antibody titer was maintained at 95% or more until sterilized twice, and the degree of contamination of general microorganisms and yeast was almost suppressed when sterilized twice. Therefore, in order to maintain the antibody titer of the yolk fluid and to prevent microbial contamination, it was confirmed that it was more effective to sterilize twice at 63 ° C. for 3 minutes (see Example 6). .

Production of Fermented Milk Using Egg Yolk Fermented milk was produced using an egg yolk liquid containing an anti-Helicobacter pylori antibody. Lactobacillus acidophilus scabii, Streptococcus thermophilus, and Bifidobacterium longum were inoculated to the sterilized raw milk at a constant concentration, cultured until the pH reached 4.5, and then cooled.
Then, a syrup was produced by dissolving the juice concentrate, dietary fiber, glucose, oligosaccharide, and the like. In particular, 63
The mixture was sterilized twice at 3 ° C. for 3 minutes, and 0.1 to 10% by weight was mixed with the culture solution and stirred. The syrup prepared in advance was mixed at a predetermined ratio, stirred and homogenized, and then packaged in a container. As a result of a sensory test of fermented milk to which egg yolk containing a specific antibody as an active ingredient was added,
Good results were shown in flavor, physical properties and overall taste (see Example 7).

Hereinafter, the present invention will be described in more detail with reference to examples. However, the following examples do not limit the scope of the present invention, and ordinary modifications can be made by those skilled in the art within the technical idea of the present invention.

Example 1 Preparation of Antigen Helicobacter pylori was cultured at 37 ° C. for 2 days in a Brucella medium containing 5% horse serum. This culture was added to 4,0
After the cells were collected by centrifugation at 00 × g, they were suspended in phosphate buffered saline. After crushing the cells with ultrasound,
After centrifugation at 10,000 × g for 20 minutes to remove unbroken cells, the collected supernatant was added to 100,100 g
After ultracentrifugation at 000 × g for 30 minutes to precipitate cell membranes, the cells were suspended in distilled water and used as antigens.

Example 2 Production of Anti-Helicobacter pylori Antibody White legphone was immunized using the outer membrane protein antigen produced in Example 1. The adjuvant is a mixture of Drakeol, a kind of mineral oil, and an emulsifier, and the emulsifier is Span 85 (Span
85) and Tween 85
The mixture was used in a ratio of 4:46. After mixing and emulsifying drakeol, an emulsifier, and an antigen in a ratio of 9: 1: 8, intramuscular injection was performed four times at two-week intervals until a sufficient antibody titer was obtained. The transition of the specific antibody titer transferred to the egg was tracked, and the egg at the stage where the antibody titer was sufficiently increased was collected to produce an egg yolk solution.

Four intramuscular injections were made at intervals. Eggs at the stage where the specific antibody titer transferred to the eggs was sufficiently increased were collected. The collected immunized eggs were separated into egg whites and yolks via a cleavage ovum, and the separated yolks were filtered with a filter, and then homogenized at 70 to 80 kg / cm2 to produce an egg yolk solution.

Example 3 Measurement of Antibody Titer Anti-Helicobacter pylori antibody titer in egg yolk was measured by the microtiter method as follows. After mixing 1 g of egg yolk solution and 9 ml of an aqueous solution of lambda carrageenan (0.15% w / v), and allowed to stand at room temperature for 30 minutes, 10,000 × g,
By centrifuging for 10 minutes, a yolk water-soluble protein fraction containing a specific antibody was obtained and used as a sample solution. The sample solution was diluted 2 n-fold with a phosphate buffer (PBS, pH 7.4) to obtain a test solution. The agglutinating antibody titer was 50 μl of each test solution and 5 μl
After mixing 0 μl with the μ plate, the mixture was left at 37 ° C. for 24 hours to examine the presence or absence of aggregation. Antibody titers were expressed as the reciprocal of the highest dilution of the sample where aggregation appeared. The antibody titer of the yolk fluid produced according to Example 2 was shown to be about 5,700.

Example 4 Antibody titer based on sugar concentration and sterilization temperature In order to enhance the storage ability of egg yolk fluid, a sterilization step is essential. However, the heat treatment conditions of this sterilization step reduce the antibody titer. Therefore, in order to prevent the antibody titer from decreasing due to the sterilization process, after adding sugar at various concentrations, the change in the antibody titer due to the sterilization temperature was confirmed as follows. First, sugar is added to the yolk solution at a concentration of 0, 10, 20, 30, or 40% by weight, and each of the yolk solutions is sterilized at 60, 63, 65, or 67 ° C. for 10 minutes using a tubular sterilizer. Was. After the sterilization treatment, a change in the antibody titer was confirmed by the method of Example 3.

As shown in FIG. 1, when 10% by weight or more of sugar is added to the egg yolk solution and treated at a sterilizing temperature of 63 ° C. or less, the antibody titer is maintained at about 80% or more. Was.

Example 5 Antibody Titer and Degree of Contamination by Sterilization Time At 63 ° C. set in Example 5, the antibody titer and the degree of contamination of general microorganisms and yeast by the sterilization time were confirmed. First, the yolk fluid was sterilized at 63 ° C. for 3, 5, 7, and 10 minutes, respectively, and then the antibody titer was measured by the method of Example 3. At the same time, 1 ml samples were taken at different sterilization times and serially diluted in decimal. 1 ml of the diluted solution was inoculated into a Petri dish, and PCA (pla
te count agar, Difco, # 0479
-17-6) After separating and consolidating the medium, the medium was cultured at 37 ° C. for 2 days. As a result, the number of colonies that appeared was counted using general microorganisms. Then, inoculate 1 ml of the diluted solution into a Petri dish and PDA
(Potato dextrose agar, Dif
co, # 0013-17-6) The medium was separated and cultured at 25 ° C. for 4 days. The number of colonies that appeared was counted by the number of yeasts.

As shown in FIG. 2, the anti-Helicobacter pylori antibody had a titer of 9 when heat-treated at 63 ° C. for up to 5 minutes.
It was shown to be maintained above 0%. However, as a result of the measurement of the degree of contamination, it was confirmed that general microorganisms and yeasts were completely killed only by heat treatment for 7 minutes or more.

Example 6 Antibody titer and degree of contamination depending on the number of sterilizations In Example 5, the sterilization conditions for maintaining the antibody titer were 63 ° C.
Although it was indicated as 5 minutes, under these sterilizing conditions, contamination of general microorganisms and yeast could not be prevented, so a method of increasing the number of sterilizations was examined. The egg yolk solution to which 10% by weight of sugar was added was sterilized up to three times under a heat treatment condition of 63 ° C. for 3 minutes, and the change in antibody titer and the degree of contamination were confirmed. As shown in FIG. 3, it was shown that the maintenance of the antibody titer was maintained as it was until the second sterilization, and the degree of contamination of general microorganisms and yeast was almost suppressed during the second sterilization. Therefore, in order to maintain the antibody titer of the yolk fluid and prevent microbial contamination, it was confirmed that it is more effective to sterilize twice for 3 minutes each time than to continuously heat-treat at 63 ° C. did.

Example 7 Production of Fermented Milk Using Egg Yolk Liquid Fermented milk was produced as shown in FIG. 4 using an egg yolk liquid containing an anti-Helicobacter pylori antibody. First, raw milk adjusted to a non-fat milk solid content of 8 to 20% by weight using skim milk powder was sterilized at 72 to 75 ° C for 15 seconds. After cooling the sterilized raw milk to a certain temperature, 1 part of Lactobacillus acidophilus, Lactobacillus scabii, Streptococcus thermophilus and Bifidobacterium longum are added.
The cells were inoculated at a concentration of 06 cfu / ml, cultured until the pH reached 4.5, and then cooled. Then, the juice concentrate 0.1
５０50% by weight, dietary fiber 0.1-20% by weight, glucose 0.5-30% by weight, oligosaccharide 0.1-15% by weight, etc. were dissolved to prepare a syrup. The syrup thus produced was sterilized and cooled. On the other hand, 10% by weight of sugar was added and the yolk liquid 0.1 sterilized twice at 63 ° C. for 3 minutes.
Was mixed with the lactic acid bacteria culture solution and stirred. The syrup prepared in advance was mixed and stirred at a predetermined ratio, homogenized, and then packaged in a container. in this way,
As a result of a sensory test of fermented milk to which egg yolk containing a specific antibody as an active ingredient was added, favorable results were shown in flavor, physical properties, and overall taste.

[0027]

As has been confirmed from the above examples, the heat treatment step of the yolk liquid according to the present invention can maintain the anti-Helicobacter pylori antibody titer as it is and suppress the contamination of general microorganisms and yeast. did it. Therefore,
ADVANTAGE OF THE INVENTION According to this invention, fermented milk can be manufactured using the yolk liquid which maintains anti- Helicobacter pylori antibody titer.

[Brief description of the drawings]

FIG. 1 is a graph showing changes in antibody titer according to sugar concentration and sterilization temperature.

FIG. 2 is a graph showing changes in antibody titer and microbial contamination with time of sterilization.

FIG. 3 is a graph showing changes in antibody titer and microbial contamination with the number of sterilizations.

FIG. 4 is a production process diagram of fermented milk using an egg yolk solution containing an anti-Helicobacter pylori antibody.

 ────────────────────────────────────────────────── ─── Continuing on the front page (72) Inventor Xu-Zu ▼ Cheng South Korea Chungcheongnam-do, Cheonan-si, Shinbuk-dong, Obayashi Hansparp 304-305 (72) Inventor Shiranaga, South Korea Seocho-gu, Seoul Seocho-gu, Seoul 1481 −14 Kenei-Biraga-dong 302 (72) Inventor Li-jeong-ri South Korea, Gyeonggi-do, Anyang-si, Dong-An-gu, Pyeongchon-dong, Prairie-Maul, Ohara Apartment 302-904 F-term (reference) 4B001 AC02 AC03 AC09 AC31 BC14 EC05 4B018 LB07 LE04 MD30 MD31 MD71 MD72 ME09 MF13

Claims (5)

[Claims] In the production of fermented milk, sugar is added to an egg yolk solution containing an anti-Helicobacter pylori antibody to maintain the antibody titer, and sterilization heat treatment is performed to prevent contamination of general microorganisms and yeast. A method for producing fermented milk, comprising mixing a sterilized egg yolk solution with a lactic acid bacteria culture solution and stirring the mixture. 2. The method for producing fermented milk according to claim 1, wherein the sugar is selected from the group consisting of sugar, high fructose, and galactooligosaccharide. 3. The method for producing fermented milk according to claim 1, wherein the content of sugar added to the egg yolk liquid is 10 to 30% by weight. 4. The method according to claim 1, wherein the sterilizing heat treatment is carried out at 60 to 65 ° C. for 2 to 1
2. The method for producing fermented milk according to claim 1, wherein the fermented milk is performed 1 to 3 times for 0 minutes. 5. The method for producing fermented milk according to claim 1, wherein the content of the heat-treated egg yolk liquid added to the lactic acid bacteria culture solution is 0.1 to 10% by weight.