KR100366302B1 - A METHOD OF PREPARING FERMENTED MILK USING EGG YOLK CONTAINING ANTI-Helicobacter pylori CHICKEN IgY - Google Patents

Abstract

The present invention relates to a method for adding an egg yolk solution containing an anti-helicobacter phyllori antibody to a conventional fermented milk process, unlike the conventional technique of adding a lactic acid bacteria growth promoting substance to an egg diluent and then inoculating the lactic acid bacteria. More specifically, it is intended to provide a sterilization method for preventing general microbial and yeast contamination as well as maintaining anti-Helicobacter pilori antibody titer during fermented milk manufacturing process.

Description

Fermented milk production method using egg yolk solution containing anti-Helicobacter pilori antibody {A METHOD OF PREPARING FERMENTED MILK USING EGG YOLK CONTAINING ANTI-Helicobacter pylori CHICKEN IgY}

The present invention relates to a method capable of maintaining titers of antibodies and preventing general microorganisms and yeast contamination when a fermented milk product is prepared using an egg yolk solution containing an anti-Helicobacter pilori antibody.

Recently, an antigen against Helicobacter pylori, known as a causative agent for gastric ulcer, has been injected into a laying hen, and studies are actively conducted using an antibody transferred from the antigen to egg yolk as an active ingredient. Japanese Patent Laid-Open No. 4-275232 discloses a food for preventing gastric ulcer and duodenal ulcer as an active ingredient of an antibody produced by using Helicobacter pylori cell lysate as an antigen. Korean Patent Publication 98-81323 discloses 0.01 to 0.1% by weight of yolk antibodies immunized against Helicobacter pylori urease and flagella, and an organism selected from lactic acid bacteria, Enterococcus, yeast and Bacillus. A pharmaceutical composition for preventing and treating gastritis, gastric ulcer and duodenal ulcer is disclosed. However, when yolk antibodies made from the above patents are used in products such as fermented milk and beverages, the titer of specific antibodies is significantly reduced by heat treatment performed in the process, thereby preventing and treating gastric ulcer, gastritis and duodenal ulcer caused by yolk antibodies. It's hard to expect an effect. In order to maintain the titer of the yolk antibody during storage, Shimizu et al. Added 30-50% by weight of sucrose or sugar for storage or maintenance of antibody titer in the process, and the temperature was 75-80 ° C. Reported heat treatment for 15 minutes (J. of food science, 1994, 59 (4), 763-772).

In 1984, Lin and Cunningham applied egg fermented milk by sterilizing 47.4% egg white, citric acid, 28.4% alkaline soybean oil, and 19.0% skim milk. Glucose (sucrose), sucrose (sucrose), xanthan gum, vanilla (vanilla) extracts were added, and then inoculated with L. bulgaricus and Streptococcus thermophilus Yogurt was developed. In 1984, Cunningham et al. Sterilized and cooled low-fat homogeneous oil, homogenized and mixed with egg white albumin (albumin) sterilized twice at 54.4 ° C, and then mixed inoculated Lactobacillus vulgaris and Streptococcus thermophilus. 6 hours at 40 ℃ to prepare a yogurt. In 1983, Kim et al. Prepared fermented milk by adding glucose, sucrose, and lactose to the homogeneous whole solution under a heat treatment condition of 58 ° C. for 30 minutes and lowering the pH to 4.8 by adding lactic acid bacteria. In the case of Japan, a patent publication (notice number: 57-54113) discloses a technique of preparing fermented milk by dissolving dry sterilized egg whites from which lysozyme of egg white is removed in milk, adding glucose, and then inoculating lactic acid bacteria. . Domestic patents include oils (notice: 97-574) sterilized by mixing lactose, sugar and egg whites with lactose, sugar, and water, and then lactobacillus ashdophyllus, Bifidobacterium longgum, and streptoco Disclosed is a method for preparing a lactic acid bacterium fermented beverage which is inoculated and mixed with Caucasus thermophilus. In the development of fermented milk using eggs as described above, fermented milk was prepared by mixing sugar, alkali-treated soybean oil, skim milk, etc. to promote fermentation in diluents of whole egg, egg white, and egg yolk, and solved physical stability such as layer separation phenomenon of fermented milk. To do this, stabilizers such as xanthan gum were used.

The present invention relates to a method for adding an egg yolk solution containing an anti-helicobacter phyllori antibody to a conventional fermented milk process, unlike the conventional technique of adding a lactic acid bacteria growth promoting substance to an egg diluent and then inoculating the lactic acid bacteria. More specifically, it is intended to provide a sterilization method for preventing general microbial and yeast contamination as well as maintaining anti-Helicobacter pilori antibody titer during fermented milk manufacturing process.

The present invention is applied to the yolk solution containing the yolk antibody immunized by the Helicobacter pylori antigen in the production of fermented milk, it provides a sterilization method that can prevent contamination of general microorganisms and yeast while maintaining the antibody titer they have It aims to do it.

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

Figure 2 is a graph of the change in antibody titer and microbial contamination according to the sterilization time.

Figure 3 is a graph of the change in antibody titer and microbial contamination according to the sterilization recovery.

Figure 4 is a manufacturing process of the fermented milk using an egg yolk solution containing an anti-helicobacter phyllori antibody.

The present inventors produced immune eggs containing antibodies from the laying hens using Helicobacter Philophyll as an antigen. Thus, only the yolk portion containing the antibody was separated from the produced immune eggs and used to prepare fermented milk. In addition, the present invention has been completed by studying sterilization conditions to prevent contamination of general microorganisms and yeasts in addition to yolk solution and fermented milk products, and to prevent reduction of antibody titers during heat treatment.

According to a preferred embodiment of the present invention, after adding the sugar to the yolk solution containing the anti-Helicobacter phyllori antibody to maintain the antibody titer and sterilization heat treatment to prevent contamination of the general microorganism and yeast after the sterilized yolk solution Fermented milk is prepared by mixing and stirring the lactic acid bacteria culture medium. Preferred sugars used to maintain antibody titers are selected from the group consisting of sugars, high fructose, galactooligosaccharides. More preferably, the sugar is sugar. The content of the sugar added to the 70 to 90% by weight yolk solution is preferably 10 to 30% by weight. In addition, the sterilization heat treatment is preferably performed once to three times at 60 to 65 ℃ for 2 to 10 minutes. According to a preferred embodiment of the present invention, it is added to the 80% by weight lactic acid culture medium and 10 to 19.9% by weight fruit syrup The content of the yolk solution is 0.1 to 10% by weight. Preparation of the antigen

Helicobacter Philosophy, known as the causative agent of gastric ulcers, was disrupted with ultrasound and centrifuged to obtain supernatant. The supernatant was ultracentrifuged to precipitate cell membranes, which were then suspended in distilled water and used as antigens (see Example 1).

Preparation of Egg Yolk Containing Anti-Helicobacter Philoly Antibody

The 40-week-old white leghorn was immunized with the envelope protein antigen. The adjuvant, emulsifier, and antigen were mixed and emulsified in a ratio of 9: 1: 8, and then injected intramuscularly four times at two-week intervals until sufficient antibodies were obtained. Eggs were harvested at which the titers of specific antibodies transferred to eggs were sufficiently elevated. The collected immune eggs were separated into egg whites and egg yolks through the harlans, and the separated egg yolks were filtered with a filter and then homogeneous at 70 to 80 kg / cm 2 to prepare egg yolk solutions. The anti-Helicobacter pilori antibody titer of the yolk solution thus prepared was about 5,700 (see Examples 2 and 3).

Antibody Titer According to Sugar Concentration and Sterilization Temperature

In order to improve the shelf life of egg yolk solution and to reduce the antibody titer, the antibody titer was changed according to sugar concentration and sterilization temperature. As a result, it was shown that treatment of 90 wt% yolk solution containing 10 wt% sugar at a sterilization temperature of 63 ° C. or less for 10 minutes can maintain the titer of the anti-Helicobacter pilori antibody at least 80% (see Example 4).

Antibody Titer and Contamination with Sterilization Time

In particular, 90 wt% yolk sac with 10 wt% sugar was sterilized at 63 ° C. for 3, 5, 7, and 10 minutes, and then the anti-Helicobacter pylori antibody titer and general microbial and yeast contamination were measured. As a result, when the annealing up to 5 minutes at 63 ℃, the antibody titer was maintained at 90% or more, and as a result of the contamination measurement, it was confirmed that the general microorganism and yeast are completely killed only by heat treatment for 7 minutes or more (Example 5).

Antibody Titer and Contamination by Sterilization Count

The 90 wt% yolk solution added with 10 wt% sugar was sterilized up to three times under heat treatment conditions of 63 ° C. for 3 minutes to confirm changes in antibody titers and contamination. As a result, the anti-H. Pylori antibody titer was maintained at 95% or more until 2 times sterilization, and the contamination of general microorganisms and yeast was also suppressed mostly in 2 times sterilization. Therefore, in order to prevent the contamination of microorganisms together with the maintenance of the antibody titer of the yolk sac solution, it was confirmed that sterilization twice in three minutes at 63 ℃ (see Example 6).

Fermented milk production using egg yolk

Fermented milk was prepared using an egg yolk solution containing anti-Helicobacter pilori antibody. Lactobacillus ashdophyllus, Lactobacillus cassia, Streptococcus thermophilus, Bifidobacterium longgum were inoculated to sterilized raw milk to incubate at a constant concentration and cooled to pH 4.5. And the syrup was prepared by dissolving the juice concentrate, dietary fiber, glucose, oligosaccharide, and the like. 90 wt% yolk solution added with 10 wt% sugar was sterilized twice at 63 ° C. for 3 minutes, and the sterilized 0.1 to 10 wt% yolk solution was cultured with 80 wt% lactic acid bacteria and 10 to 19.9 wt% prepared above. Stir with% syrup. Here, the pre-prepared syrup was mixed, stirred and homogenized at a predetermined ratio, and then packed in a container. As a result of sensory evaluation of fermented milk containing egg yolk containing a specific antibody as an active ingredient, the result was satisfactory in flavor, physical properties and overall taste (see Example 7).

The present invention will be described in more detail with reference to the following Examples. However, the following examples are not intended to limit the scope of the present invention, and ordinary changes by those skilled in the art are possible within the scope of the technical idea of the present invention.

Example 1

Preparation of the antigen

Helicobacter pylori was incubated for 2 days at 37 ° C. in Brucella medium containing 5% horse serum. The culture was centrifuged at 4,000 × g to recover the cells and then suspended in phosphate buffered saline. The cells were crushed by ultrasound, centrifuged at 10,000 × g for 20 minutes to remove the unbroken cells, and the recovered supernatants were ultracentrifuged at 100,000 × g for 30 minutes to precipitate cell membranes, which were then suspended in distilled water and used as antigens. .

Example 2

Preparation of Anti Helicobacter Philoly Antibody

The white leghorn was immunized using the outer membrane protein antigen prepared in Example 1. The adjuvant was used by mixing Drakeol, which is a kind of mineral oil, with an emulsifier, and using Span 85 and Tween 85 at a ratio of 54:46. Drakeol, emulsifier, and antigen were mixed and emulsified in a ratio of 9: 1: 8, and then intramuscularly injected four times at two week intervals until sufficient antibody titer was obtained. The yolk solution was prepared by tracking the specific antibody titer transition to the egg and collecting the eggs at the stage where the antibody titer was sufficiently elevated.

Four intramuscular injections were made at intervals. Eggs were harvested at which the titer of the specific antibody transferred to the eggs was sufficiently elevated. The collected immune eggs were separated into egg whites and egg yolks through the harlans, and the separated egg yolks were filtered with a filter and then homogeneous at 70 to 80 kg / cm 2 to prepare egg yolk solutions.

Example 3

Antibody titration

Anti-helicobacter phyllori antibody titer in egg yolk was measured by the microtiter method as follows. 1 g of egg yolk solution and 9 ml of lambda carrageenan solution (0.15% w / v) were mixed and left at room temperature for 30 minutes, followed by centrifugation at 10,000 × g for 10 minutes to obtain an aqueous yolk soluble protein fraction containing a specific antibody. . The sample solution was diluted 2n times with phosphate buffer (PBS, pH 7.4) to prepare a sample solution. Agglutinated antibody titer is obtained by mixing 50 μl of each sample solution and 50 μl of bacterial crushed solution on a microplate, and then standing at 37 ° C. for 24 hours to investigate the presence of aggregation. Antibody titers were expressed as the inverse of the maximum dilution factor of the sample in which aggregation was seen. The antibody titer of the yolk solution prepared by Example 2 was about 5,700.

Example 4

Antibody Titer According to Sugar Concentration and Sterilization Temperature

Sterilization is essential to increase shelf life of yolk solution. However, due to the heat treatment conditions of the sterilization process, the titer of the antibody may decrease. Therefore, in order to prevent the decrease in antibody titer according to the sterilization process, after changing the sugar titer at various concentrations, the change in antibody titer according to the sterilization temperature was confirmed as follows. First, sugar is added to 100, 90, 80, 70, and 60% by weight of egg yolk solution at concentrations of 0, 10, 20, 30, and 40% by weight, respectively, and each yolk solution is prepared using a tubular type sterilizer. Sterilized at 65 ° C, 67 ° C for 10 minutes. After the sterilization treatment, the change in antibody titer was confirmed according to the method of Example 3.

As shown in Fig. 1, the antibody titer was maintained at about 80% or more when more than 10% by weight of sugar was added to less than 90% by weight of yolk solution and treated at a sterilization temperature of 63 ° C or lower.

Example 5

Antibody Titer and Contamination with Sterilization Time

The antibody titer and the degree of contamination of the general microorganism and the yeast according to the sterilization time at 63 ° C. set in Example 5 were confirmed. First, the yolk solution was sterilized at 63 ° C. for 3, 5, 7, and 10 minutes, and antibody titers were measured according to the method of Example 3. At the same time, 1 ml of the sterilized samples were taken and serially diluted. 1 ml of the dilution solution was inoculated in a Petri dish, and the PCA (plate count agar, Difco, # 0479-17) medium was dispensed and hardened, followed by incubation for 2 days at 37 ° C. 1 ml of the diluted solution was inoculated in a petri dish, and the PDA (potato dextrose agar, Difco, # 0013-17-6) medium was dispensed and cultured at 25 ° C. for 4 days to count the number of colonies as yeast water.

As shown in Fig. 2, when heat treated at 63 ° C. for 5 minutes, the titer of the anti-Helicobacter pilori antibody was maintained at 90% or more. However, it was confirmed that general microorganisms and yeast were completely killed only by heat treatment for more than 7 minutes.

Example 6

Antibody Titer and Contamination by Sterilization Count

In Example 5, the sterilization condition for maintaining the titer of the antibody was 63 ℃, 5 minutes, but the sterilization conditions can not prevent the contamination of the general microorganism and yeast, the method to increase the number of sterilization was examined. 90 wt% of the yolk solution with 10% by weight of sugar was sterilized up to three times under heat treatment conditions of 63 ° C. for 3 minutes to confirm changes in antibody titers and contamination. As shown in Figure 3, the maintenance of the antibody titer was maintained as it is until two sterilization, the contamination of the general microorganism and yeast also appeared to be mostly suppressed in two sterilization. Therefore, in order to prevent the contamination of the microorganism with the maintenance of the antibody titer of the yolk solution, it was confirmed that sterilization twice over three minutes than the continuous heat treatment at 63 ℃.

Example 7

Fermented milk production using egg yolk

Fermented milk was prepared as shown in Fig. 4 using an egg yolk solution containing an anti-Helicobacter pilori antibody. First, raw milk with a nonfat milk content adjusted to 8.5% by weight using skim milk powder was sterilized at 75 ° C. for 15 seconds. After cooling the sterilized raw oil to a certain temperature, inoculating Lactobacillus ashdophyllus, Lactobacillus cassia, Streptocogus thermophilus, Bifidobacterium longgum at a concentration of 106 cfu / ml until pH 4.5 After incubation it was cooled. And the juice concentrate 1.0% by weight, dietary fiber 0.8% by weight, glucose 7% by weight, oligosaccharides 6% by weight, etc. dissolved in 85.2% by weight of water to prepare a syrup. The syrup thus prepared was sterilized and then cooled. On the other hand, 90% by weight of the yolk solution to which 10% by weight of sugar was added was sterilized twice at 63 ° C for 3 minutes. 1.0% by weight of the sterilized yolk solution was mixed with the 80% by weight of the lactic acid bacteria culture medium and 19.0% by weight of the syrup prepared in advance, homogenized by stirring, and then packaged in a container. As a result of sensory evaluation of fermented milk containing egg yolk containing a specific antibody as an active ingredient, the results were satisfactory in flavor, physical properties and overall taste.

As confirmed in the above embodiments, the heat treatment process of the yolk solution according to the present invention was able to suppress contamination of general microorganisms and yeast while maintaining the titer of the anti-Helicobacter pilori antibody. Therefore, according to the present invention, fermented milk can be prepared using an egg yolk solution that maintains the anti-Helicobacter pilori antibody titer.

Claims (5)

In the fermented milk manufacturing method, Maintaining the antibody titer by adding a sugar having a weight ratio of 10 to 30% by weight selected from the group consisting of sugar, high fructose and galactooligosaccharides to 70 to 90% by weight yolk solution containing an anti-Helicobacter pilori antibody; Preventing the contamination of the microorganisms and yeasts in which the sugar-added yolk solution is sterilized by one to three times at 60 to 65 ° C. for 2 to 10 minutes; Fermented milk production method comprising the step of mixing and stirring the sterilized yolk solution in the lactic acid bacteria culture solution and syrup. delete delete delete The method of claim 1, wherein the mixed lactic acid bacteria culture solution is stirred at 80% by weight, the syrup is 10-19.9% by weight, and the sterilized egg yolk solution added is 0.1-10% by weight. .