JP4005103B2 - Bactericidal beverage containing unmodified lactoferrin and method for producing the same - Google Patents

Description

  The present invention relates to a sterilized beverage containing unmodified lactoferrin, which is heat-sterilized and mixed with unmodified lactoferrin in a sterilized beverage material solution (excluding soymilk material solution), and a method for producing the same. Specifically, the present invention relates to a sterilized beverage in which the contained lactoferrin is not inactivated by heat despite being heat sterilized, and a method for producing the same.

  In the present invention, beverages mean normal beverages, excluding soy milk, and examples include fruit beverages, dairy beverages, vegetable beverages, coffee, various teas, cocoa, etc. in addition to milk. Can do.

  In general, various beverages are commercially available. However, such beverages are usually strictly sterilized by heat in order to ensure storage and safety as foods. It is distributed in the market in a sealed state.

  However, a sterilized beverage containing sterilized native lactoferrin is not known and there is no literature.

  Lactoferrin is an iron-binding protein contained in tears, saliva, peripheral blood, milk, and the like in vivo, and has an antibacterial action against harmful bacteria [Non-Patent Document 1], an action to promote absorption of iron from the intestinal tract [non- Patent document 2], immunostimulatory action [Non-patent document 3], cell proliferation action [Non-patent document 4], anti-inflammatory action [Non-patent document 5], bifidobacteria growth effect [Patent document 1, non-patent document 6, non-patent document Patent Document 7] and the like have been reported to have various physiological activities.

  In addition, lactoferrin is blended in various foods, processed foods, pharmaceuticals, etc. In recent years, infant milk containing lactoferrin has been released.

  Conventionally, it is known that lactoferrin is neutral and thermally unstable, and is almost deactivated by heating at 62.5 ° C. for 30 minutes and completely inactivated by heating at 70 ° C. for 15 minutes [non- Patent Document 8], by adjusting the pH of a lactoferrin solution to 1.0 to 6.5 with an acid in advance, the heat stability of lactoferrin is increased, and the maximum heating temperature is 130 ° C. for 1 to 2 minutes. It is disclosed that the native rate of lactoferrin is 60% or more (Patent Document 2).

  However, in order to provide beverages to consumers safely, it is necessary to kill bacteria derived from the raw materials, and for example, heat treatment is performed at 130 ° C. for 2 seconds or more.

  In addition, for the purpose of long-term storage, it is necessary to completely kill heat-resistant spore bacteria in addition to general bacteria, but when heat-treated at a temperature of 100 to 130 ° C., thermophilic bacteria form heat-resistant spores. In some cases, the thermospores may germinate and proliferate during storage.

  Therefore, in order to produce a sterilized beverage that can be stored for a long period of time, F0 = 3.1 (Z, which is the minimum sterilization condition that can kill heat-resistant spore bacteria in the beverage and completely sterilize Clostridium boturinum. It is necessary to sterilize under heating conditions satisfying above.

  F0 is a lethal value of heat sterilization, and means the heating time (minute) required to kill a certain number of bacteria at a constant temperature, and Z shortens the heat killing time of microorganisms to 1/10. The temperature increase of Fahrenheit required for this process is shown. The larger this value, the higher the heat resistance of the microorganism (Non-patent Document 9).

  In any case, when heat treatment is performed for the purpose of sterilization as described above, the activity of lactoferrin is lost by heat.

  Moreover, it is also known that a lactoferrin degradation product has a bifidobacteria growth promotion effect, a bacteriostatic bacteriostatic effect, and a bactericidal effect like lactoferrin (for example, Non-Patent Document 10, Patent Document 3 and the like).

  The present inventors previously filed a patent application for unmodified lactoferrin-containing sterilized soymilk and a method for producing the same (Japanese Patent Application No. 11-17519, hereinafter referred to as a prior application). It is confirmed that the present invention can be applied to beverages, and the present invention is applied for a patent.

  On the other hand, the term “milk” as defined in the Ministerial Ordinance on Milk and Dairy Product Component Standards (December 27, 1951, Ministry of Health and Welfare No. 52, hereinafter referred to as the Ministry of Milk Ordinance) is also broadly defined. In some cases, it may be distributed as a kind of beverage or beverage ingredient.

  As described above, since lactoferrin is an iron-binding protein contained in milk or the like, “milk or the like” circulated as a beverage (in the present invention, “milk or the like” circulated as a beverage or beverage ingredient is milked. However, when a consumer drinks this, various physiological effects of lactoferrin contained in “milk” are naturally expected. From such a point of view, research has been continued on lactoferrin contained in milk. For example, Patent Document 4 discloses a technique for separating lactoferrin from whey or skim milk.

However, in milk, when it is distributed as a beverage as described above, it is the same as other beverages that a strict heat sterilization treatment is required, and at the stage of distribution to the market as a beverage product, the activity of the contained lactoferrin Is lost by heating.
Japanese Patent Publication No. 7-79684 Japanese Patent No. 2688098 JP-A-5-320068 JP 63-152400 A Journal of Dairy Science, Volume 67, Page 3, 1984 Journal of Pediatric Gastroenterology and Nutition, Volume 2, page 693, 1983 Kunio Yamauchi, Noriaki Imamura, Tetsuro Morita, “Characteristics and Health of Milk Ingredients”, Table IV-7, page 101, Koseikan, 1993 Shimamura et al., “Lactoferrin stracture and Function”, 1st International Lactoferrin Symposium, Hawaii, 1992 Biochimica et Biophysica Acta, 715, 116, 1982 Journal of Japanese Society of Pediatrics, Vol.87, No.6, pp. 1000-1013, 1983 Pediatric Research, Vol. 29, No. 2, pp. 208-213, 1991 Journal of Pediatrics, 90, 29, 1977 Dairy technology course editorial committee edition, "dairy technology course volume 1 milk", pp. 250-252, Asakura Shoten, 1963 Journal of Japanese Society of Nutrition and Food, Vol.42, No.1, pp. 13-19, 1989

  In view of the prior art, the present inventors, as a result of intensive research, in milk, once separated lactoferrin solution from raw milk, only the separated lactoferrin solution, heat sterilized under special conditions that do not denature lactoferrin, In addition, other ingredients are heat-sterilized by a conventional method, and each is mixed again after sterilization, so that heat-sterilized milk that does not substantially adjust the ingredients despite containing unmodified lactoferrin can be obtained. I found it.

  In addition, in the case of a general beverage without being limited to this, without sacrificing the physiological activity of lactoferrin by aseptically adding an unmodified lactoferrin solution separately sterilized to the sterilized beverage raw material liquid, The present inventors have found that a sterilized beverage containing unmodified lactoferrin at a ratio of 0.02 to 5.0% (mass) and that sterilized beverage can be produced.

  The present invention aims to improve the functionality of beverages by adding unmodified lactoferrin having various physiological effects or preventing inactivation during heat treatment of lactoferrin. Bactericidal beverage containing unmodified lactoferrin consisting of heat-sterilized milk that retains its effect as it is, bactericidal beverage containing native lactoferrin that retains the bifidobacteria growth effect of lactoferrin, and the bifido growth effect of lactulose and the like in addition to lactoferrin Thus, an object of the present invention is to provide an unmodified lactoferrin-containing sterilized beverage that further improves the improvement of the intestinal flora, and a method for producing the same.

The first invention of the present invention that solves the above-mentioned problems is a pasteurized beverage raw material solution (excluding soymilk raw material solution) containing aloe extract and lactulose mixed with heat-sterilized and unmodified lactoferrin. A sterilized beverage containing unmodified lactoferrin. Moreover, 1st invention of this invention contains heat-sterilized and undenatured lactoferrin in the ratio of 0.02-5.0% (mass), and heat-sterilized lactoferrin is 80- It is desirable that the activity remains at a rate of 100% (mass).

The second invention of the present invention for solving the above-mentioned problems is the following steps f) to h):
f) A step of sterilizing and cooling a raw material liquid for beverages (excluding the raw material liquid for soy milk) containing aloe extract and lactulose ,
g) A step of adding an acid to the lactoferrin solution to adjust the solution to acidity and heat sterilization; and h) 0.02-5. Adding at a rate of 0% (mass);
A method for producing a sterilized beverage containing unmodified lactoferrin, comprising:

  In the second invention, the heat-sterilized lactoferrin remains active at a rate of 80 to 100% (mass), and the heat-sterilization of the lactoferrin solution is 2 to 5 at a temperature of 150 to 155 ° C. It is desirable that the heating is performed for a second and that the lactoferrin solution is adjusted to an acidity in the range of pH 2.0 to 5.5.

The present invention is a sterilized beverage containing unmodified lactoferrin in which a sterilized beverage raw material liquid (excluding soymilk raw material liquid) is heat-sterilized and mixed with unmodified lactoferrin, and a method for producing the same. The effects that can be achieved are as follows.
1) Improve the bacterial composition of human or animal abnormal intestinal flora by synergistic effects of native lactoferrin and lactulose bifidobacterial growth factor.
2) Since the soft drink containing unmodified lactoferrin of the present invention having the above-mentioned effects is heat-sterilized, it can be stored for a long period of time and can be immediately drunk from a container.
3) The sterilized beverage containing unmodified lactoferrin of the present invention does not deteriorate in quality due to storage.

[Reference invention]
The reference invention of the present invention is that the raw material liquid is milk, the heat-sterilized, and the unmodified lactoferrin is separated from the lactoferrin contained in the milk and separately heat-sterilized, It is.

Further, another reference invention of the present invention includes the following steps a) to e):
a) separating cream from milk to obtain skim milk;
b) a step of separating lactoferrin from the obtained skim milk;
c) a step of heat-sterilizing the separated lactoferrin under acidic conditions,
d) a step of heat sterilizing the cream separated in a) and / or b) after lactoferrin separation, and e) a heat sterilized cream and / or skim milk, and c ) And heat-sterilized unmodified lactoferrin, and preparing the mixed sterilized milk as a sterilized beverage containing unmodified lactoferrin,
A method for producing a sterilized beverage containing unmodified lactoferrin, comprising:

These reference inventions have the following effects. That is, 4) When the present invention is applied to milk, there is an effect that heat-sterilized milk in which lactoferrin is not denatured can be obtained while being heat-sterilized milk in which components are not substantially adjusted.
[Invention]
Next, the present invention will be described in detail.

The first invention of the present invention is a pasteurized paste containing unmodified lactoferrin in which a pasteurized beverage material solution (excluding soymilk solution) containing aloe extract and lactulose is heat-sterilized and mixed with unmodified lactoferrin. Beverages.

  The beverage of the present invention can be obtained by sterilizing a beverage raw material and then mixing sterilized lactoferrin separately from the raw material under aseptic conditions. In this case, as a method of sterilizing lactoferrin, for example, by adopting the technique described in Patent Document 2, the sterilization treatment can be performed without modifying the lactoferrin.

  Next, preferred aspects of the first invention of the present invention will be described. In order to facilitate understanding of the desirable aspects, the reference invention and the second invention of the present invention will be described first, and then the first invention will be described again. The invention will be described.

  The reference invention is a manufacturing method exclusively for milk as a beverage. The starting material in this case is milk, but here milk is milk, special milk, sterilized goat milk, partially skimmed milk, skimmed milk, processed milk, etc. It is meant to be used exclusively for drinking or for drinking ingredients.

  First, the cream is separated from the milk to obtain skim milk. The method for separating the cream is a known method, and an ordinary centrifugal separator (such as a cream separator) can be used.

  Next, lactoferrin is separated from the skim milk after separating the cream. As a method for separating lactoferrin, for example, the method disclosed in Patent Document 4 can be used.

  That is, first, skimmed milk is applied to a weakly acidic cation exchanger having a carboxymethyl group and a hemoglobin adsorption capacity of 3.5 g / 100 ml or more at a temperature of 0 to 60 ° C., preferably 0 to 10 ° C. Separating lactoferrin by contacting and adsorbing, washing the adsorbed weakly acidic cation exchanger with water, and desorbing the washed weakly acidic cation exchanger with a salt solution (preferably an aqueous solution of salts). Can do.

  In this case, the weak acid cation exchanger has a volume change rate of 1.5 or less (however, the volume change rate is a bed volume when the exchanger in Na form is swollen with water). Is preferably divided by the bed volume when the exchanger is equilibrated with a sodium chloride solution having an ionic strength of 0.5 or less), and the salt solution includes sodium chloride, potassium chloride, calcium chloride, Desirably, the salt solution is selected from the group consisting of magnesium chloride and mixtures thereof.

  The desorption treatment step includes a first desorption treatment step in which the weakly acidic cation exchanger is treated with a relatively low concentration aqueous solution of salts to remove components desorbed from the ion exchanger; It comprises a second desorption treatment step in which the cation exchanger is treated with a relatively high concentration aqueous solution of salts to remove components desorbed from the ion exchanger, and lactoferrin is 98% or more of the total protein recovered. It is desirable to obtain with a purity of In this case, the concentration of the relatively low concentration aqueous solution is preferably 0.4 to 2.5% by mass, and the concentration of the relatively high concentration aqueous solution is preferably 1.5 to 12% by mass.

  Thus, if the technique disclosed in Patent Document 4 is used, there is an advantage that the skim milk after separating the lactoferrin is not damaged at all and can be processed in a large amount.

  The lactoferrin separated as described above is heat sterilized by a method that does not denature, and as such a method, it is desirable to employ the technique disclosed in Patent Document 2. That is, the pH of the lactoferrin solution is adjusted to acidity in the range of 1.0 to 6.5, preferably pH 2.0 to 5.5, and heat-treated at a temperature of 60 ° C. or higher.

  Examples of the acid used for pH adjustment include lactic acid, hydrochloric acid, acetic acid, and citric acid, but lactic acid is particularly desirable from the viewpoint of flavor.

  The lactoferrin solution whose pH is adjusted is sterilized by a known method, for example, by heating at a temperature of 150 to 155 ° C. for 2 to 5 seconds. As is apparent from the test examples described later, by adjusting the pH to the above range, 80% or more of the lactoferrin after heat sterilization remains unmodified and maintains physiological activity.

  On the other hand, the cream separated from the milk in the first step and the skim milk after separating the lactoferrin are heat-sterilized by a conventional method. In this case, it is preferable to sterilize by heating after mixing the cream and skim milk again, but it is also possible to sterilize each separately.

  Finally, the native lactoferrin after heat sterilization is mixed with the cream and skim milk. This operation is preferably performed under aseptic conditions. Moreover, it is desirable to consider the mixing ratio so that the components after mixing are the same components as the first raw milk. After mixing, the container can be filled and sealed by a known method to obtain a final product.

  When the beverage is pasteurized skim milk, the heat treatment and mixing of the separated cream is unnecessary, and only the pasteurized skim milk and the heat-sterilized unmodified lactoferrin are mixed. good. That is, as described above, the separated cream and skim milk after lactoferrin separation are heat sterilized, and the heat-sterilized cream and skim milk are separated from the skim milk and heat-sterilized unmodified lactoferrin. In addition to the aspect of mixing the lactoferrin, only the skim milk after lactoferrin separation is heat sterilized, and the aspect of mixing the non-denatured lactoferrin separated from the skim milk and heat sterilized only to the heat sterilized skim milk is also adopted. It can be done.

  The pasteurized milk obtained through each of the steps as described above is one in which lactoferrin has not been deactivated while being in a state substantially free of components from the milk before pasteurization, in other words, the reference invention. By the production method, sterilized milk containing unmodified lactoferrin, which is a reference invention of a sterilized beverage containing unmodified lactoferrin, can be obtained.

  In addition, if supplemented, when lactate is added to lactoferrin separated from skim milk to adjust pH, such lactic acid is obtained by adding a starter to raw skim milk and fermenting. Can be used. That is, before or after separating the lactoferrin, the skim milk is sterilized and cooled, and after adding a starter and fermented to prepare fermented milk, the lactic acid of the obtained fermented milk is used. . Since such lactic acid is derived from raw material milk, the sterilized milk finally obtained is composed of only components derived from raw material milk, and is more preferable.

  Next, the second invention of the present invention will be described.

  The beverage in the method of the second invention of the present invention is not limited to milk, and the raw material liquid is a raw material liquid that can produce a normal beverage.

  For example, if it is a fruit drink, the raw material liquid of a drink can be prepared by mixing thawed frozen concentrated fruit juice, sugar, acidulant, flavor, colorant and other raw materials in raw water. Moreover, if it is a dairy drink, especially a lactic acid bacteria drink, skim milk can be sterilized and cooled, a starter is added and fermented, fermented milk can be prepared, and the raw material liquid of a drink can be manufactured. Moreover, if it is a coffee drink, the raw material liquid of a drink can be prepared by mixing and stirring liquid sugar and powdered milk solution to the coffee pouring liquid. Needless to say, milk may be used as a beverage raw material liquid.

The method of the present invention is characterized in that the beverage raw material liquid contains aloe extract and lactulose .

  The aloe extract is, for example, aloe plant leaf flesh such as cape aloe, aloe vera, kidachi aloe, or so-called "aloe" or "aloe gel" obtained by squeezing the leaves, and powdered "aloe powder" ”, Refined“ Aloe extract ”,“ Aloe extract ”, and the like, which include all raw materials that provide an active ingredient derived from an aloe plant, and the shape, the degree of processing or the type thereof are not limited. However, in the present invention, it is desirable to use raw materials that are commercially available for the purpose of pharmaceutical raw materials, food raw materials, and the like, and it is recommended to use a liquid obtained by concentrating aloe juice.

  In the following description, when an aloe juice concentrate is used as the aloe extract, the term “aloe extract” is followed by the concentration factor. That is, when it is simply expressed as “aloe extract”, it is a term encompassing all the raw materials related to aloe as described above, but when it is expressed as “aloe extract (5-fold concentrated)”, it is aloe juice. Means a concentrated liquid (liquid concentrated 5 times). The latter notation method is used in the examples described later.

  In the present invention, the aloe extract as described above is preferably contained in the beverage raw material liquid in the range of 0.5 to 20.0% by mass.

  Lactulose (4-O-β-D-galactopyranosyl-D-fructofuranose) is a known substance as a bifidobacterial growth-promoting factor, and is not naturally produced by carrying out the Lobli-Drewryin rearrangement of lactose It is a disaccharide. Since this lactulose has high solubility in water, it is difficult to obtain a stable powder, and it is usually used in the form of syrup.

  In general, lactulose syrup contains 45-55% lactulose, 2-8% galactose, 2-5% lactose, and 2-8% other sugars in addition to moisture. The purity is 70-90%. In addition, as a lactulose product, a product that has been powdered or crystallized in a complicated process is also commercially available.

  In the present invention, such lactulose is desirably contained in the beverage raw material liquid in the range of 0.5 to 4.0% by mass.

  Inositol is a type of vitamin B group that is abundant in rice bran, and most of the world's raw material for producing inositol is rice bran. Inositol was isolated from cereals in 1850, and nine types of cubic isomers have been recognized, but only myo-inositol is present in the animal and plant kingdoms and exhibits physiological activity. Myo-inositol is free in the living body and widely distributed in the brain, spinal cord, heart, kidney, thyroid, eyeball, muscle, testicle, etc., and plays an important role in the cells of each tissue.

  Inositol has about 50% sweetness of sucrose and is stable to heat, light, acid and alkali, and has been conventionally used for growth promotion, prevention of fatty liver and cirrhosis, arteriosclerosis prevention, aging prevention, etc. Pharmacological effects are expected, and future potential as a health food is promised (The above description regarding inositol is Japan Food Science, Vol. 24, No. 3, pages 73-80, 1985. by.).

  In the present invention, it is desirable to contain such inositol in the beverage raw material liquid in the range of 0.001 to 1.0% by mass.

As described above, in the present invention, first, aloe extract and lactulose are selected and contained in the beverage raw material liquid to prepare.

  The prepared beverage raw material liquid is sterilized by a known method (for example, UHT sterilization method, etc.), and is homogenized by, for example, a homogenizer.

  The lactoferrin in the second invention of the present invention may be a commercially available product, and can be prepared from milk by a known method (for example, the method described in Patent Document 4) and used. Although lactoferrin is usually cheaply separated from milk, it is not limited to lactoferrin derived from milk. The amount of lactoferrin added to the beverage is 0.02% or more with respect to the beverage, as is apparent from the test examples described later, and is preferably in the range of 1.0 to 5.0%.

  The lactoferrin degradation product in the second invention of the present invention may be a commercially available product, or can be prepared from bovine lactoferrin by a known method (for example, the method described in Patent Document 3) and used. A lactoferrin degradation product is usually obtained from lactoferrin separated from milk, but is inexpensive, but is not limited to lactoferrin degradation products derived from milk in the present invention.

  In the second invention of the present invention, this lactoferrin degradation product can also be added to a beverage in combination with lactoferrin. The addition amount of the lactoferrin degradation product is preferably within the range of the lactoferrin addition ratio. Specifically, when the unmodified lactoferrin and the lactoferrin degradation product are added in combination, the total addition amount is 0.02% or more, preferably 1.0 to 5.0%.

  The lactoferrin solution is prepared as follows. That is, lactoferrin alone or lactoferrin and a lactoferrin degradation product are dissolved in water at a concentration of 10 to 25%, and an acid is added to adjust the pH to a range of 2.0 to 5.5. Examples of the acid used for pH adjustment include lactic acid, hydrochloric acid, acetic acid, and citric acid, but lactic acid is particularly desirable from the viewpoint of flavor.

  The lactoferrin solution whose pH is adjusted is sterilized by a known method, for example, by heating at a temperature of 150 to 155 ° C. for 2 to 5 seconds. As is apparent from the test examples described later, 80% or more of the lactoferrin after heat sterilization remains unmodified by adjusting the pH to the above range. Therefore, in the method of the present invention, the physiological activity of lactoferrin is maintained despite heat sterilization.

  Next, a predetermined amount of the heat-sterilized unmodified lactoferrin solution is added to the sterilized beverage, mixed uniformly, and aseptically filled in a container by a known method, and sealed to obtain a final product.

  In addition, vitamins, fruit juices, fragrances, and the like can be added to the sterilized beverage containing unmodified lactoferrin as necessary. Examples of vitamins to be added include vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin C, vitamin D, vitamin E, niacin, pantothenic acid, folic acid, vitamin K, and β-carotene. Moreover, these mixtures can also be used suitably. Moreover, as fruit juice, apple juice, mandarin orange juice, banana juice, etc. can be used suitably.

  Furthermore, as a fragrance | flavor, vanilla flavor, a yogurt flavor, etc. can be used suitably.

  The first invention of the present invention is an unmodified lactoferrin-containing sterilized beverage that is preferably manufactured by the manufacturing method as described above.

  The unmodified lactoferrin-containing sterilized beverage of the present invention is heat-sterilized at least in accordance with the provisions of the Food Sanitation Law, so that it can be easily stored and transported and can be immediately used for drinking. As described above, even when stored for a long period of time, the production of aggregated precipitates of lactoferrin is not generated, and a good quality that is the same as that immediately after production is maintained.

  In addition, the germicidal beverage containing unmodified lactoferrin of the present invention is, as will be apparent from the following test examples, due to the synergistic effect of the bifidobacteria growth effect of lactoferrin and the bifidobacteria growth effect contained in the beverage, It has an excellent effect of further improving the improvement. Moreover, this effect is more remarkably exhibited by using a lactoferrin degradation product in combination.

  Furthermore, the sterilized beverage containing unmodified lactoferrin of the present invention can be applied to the field of milk. In this case, the lactoferrin is in an unmodified state of lactoferrin originally contained in milk as a raw material liquid. It can be sterilized with.

  That is, the raw material liquid is milk, and lactoferrin is separated from the milk before sterilization and separately heat sterilized, and heat sterilized and unmodified lactoferrin is sterilized by mixing the milk again before sterilization. It will have substantially the same component composition as milk, in other words, it will be a sterilized milk with almost no component adjustment.

  In the present invention described above, the heat sterilization conditions are such that the heat-resistant spore bacteria can be completely killed and stored for a long time, for example, F0 = 3.1 (Z = 18) or more. It is desirable that the heating conditions satisfy the conditions, specifically, the conditions of 150 to 155 ° C. for 2 to 5 seconds. Thus, if the present invention is applied to a sterilized beverage that can be stored for a long period of time, the effects of the present invention can be enjoyed to the maximum.

Next, a test example is shown and this invention is demonstrated in detail.
Test example 1
This test was conducted to examine the remaining lactoferrin activity by heating.
1) Sample preparation Commercially available lactoferrin (manufactured by Oleophyna, Belgium) was dissolved in purified water at a concentration of 1%, 90% first lactic acid (manufactured by Nacalai Tesque) was added, and pH was expressed. The value shown in 1 was adjusted.
2) Test method The pH-adjusted lactoferrin solution is divided into two equal parts, one is not heated, and the other is heated directly at 155 ° C. for 2 seconds (F 0 = 81, Z = 18) using a direct heating sterilizer (manufactured by Morinaga Milk Industry). And then cooled to room temperature.

For each sample cooled to room temperature, the residual amount of lactoferrin was measured by the rocket method [Analical Biochemistry, Vol. 15, pp. 45-52, 1966]. The residual rate (%) was calculated from the ratio of the residual amount of lactoferrin in each heated sample to the residual amount.
3) Test results The results of this test are as shown in Table 1. As is clear from Table 1, the lactoferrin solution adjusted to a pH in the range of 2.0 to 5.5 had a residual rate of 80% or more when heat-treated at 155 ° C. for 2 seconds.

  From this test result, it was found that even when a lactoferrin solution having a pH of 2.0 to 5.5 was heated at a high temperature, the lactoferrin activity remained at a high rate, that is, unmodified lactoferrin was present.

  In addition, although it tested by changing the density | concentration of lactoferrin and the lactoferrin, pH, and heating temperature, the substantially same result was obtained.

Test example 2
This test was conducted to examine the effect of the sterilized beverage containing the native lactoferrin of the present invention.
1) Preparation of sample (1) Preparation of administration feed A sterilized beverage containing unmodified lactoferrin was prepared by the same method as in Example 1.
(2) Test animals Abnormalities similar to the initial infection time of newborns and intestinal flora of premature infants by Tanaka's method (Therapeutics, Vol. 14, No. 5, 594-597, 1985) Mice that stably retained the intestinal floor were prepared.
2) Test method Ten 5-week-old SPF mice (SLC) Balb / c females prepared by the method described above were divided into two groups of five each and placed in a cage containing a fecal feeding prevention net. One group (control) ) Was bred for 1 week on a normal solid food, and then bred for 1 week with free intake of only milk. The other group was bred for 1 week on a normal regular solid food, and then was bred for 1 week with free intake of the sterilized beverage containing the unmodified lactoferrin. Both groups received water freely.
After administration of milk and a non-denatured lactoferrin-containing sterilized beverage, fresh stool from each mouse on days 1, 3, 5 and 7 was collected over time, and Mitsuoka's method ("World of Enteric Bacteria", Sobunsha, 1980). The number of harmful bacteria in feces was measured by the following method, and the average value of each group was calculated and tested.

3) Test results As a result, the number of harmful bacteria such as Enterobacteriaceae, Streptococcus, Staphylococcus, and Clostridium in feces over time after administration of each sample was 9.0 ± 0.2 (No. 1st day), 8.2 ± 0.2 (3rd day), 8.1 ± 0.2 (5th day), 9.8 ± 0.9 (7th day. Detection rate 60% )Met.

  In contrast, in the bactericidal beverage administration group containing unmodified lactoferrin, the logarithmic values were 6.2 ± 0.2 (first day), 6.1 ± 0.2 (third day), and 5.2, respectively. It was below ± 0.3 (5th day), 7.0 (7th day, detection limit) or less.

  In addition, the number of these harmful bacteria in the stool was maintained almost stably over several weeks unless the breeding conditions were changed. From these results, the number of harmful bacteria in mice bred with the unmodified lactoferrin-containing sterilized beverage of the present invention is reduced to 100-1000 or less compared with that of mice bred with milk, and more than twice as many abnormal It has become possible to produce a mouse that stably retains the intestinal flora for a long period of time.

Test example 3
This test was conducted in order to examine the effect of the addition amount of native lactoferrin.
1) Sample preparation Bovine lactoferrin in proportions of 0, 0.02, 0.04, 0.06, 0.1, 0.5, 1.0, 2.0, 5.0, and 7.0% Ten samples were prepared by the same method as in Example 1 except that it was added to the beverage.
2) Test Method 50 mice prepared by the same method as in Test Example 2 were divided into 10 groups of 5 mice, and the test was performed by the same method as Test Example 2.
3) Test results As a result of this test, the number of harmful bacteria in feces was reduced by administration of 0.02% or more of the unmodified lactoferrin-added sample, and was stably maintained at a low level after 3 days of administration. This effect became more prominent as the amount of unmodified lactoferrin added was increased. In addition, the number of bacteria of clostridial in stool of the 1.0-5.0% native lactoferrin added sample administration group decreased below the detection limit, and in the 7.0% native lactoferrin added sample administration group, 5.0. No difference was observed from the group administered with a sample containing 1% unmodified lactoferrin.

  From this result, the addition of 0.02% or more, preferably 1.0-5.0% unmodified lactoferrin reduces the number of harmful bacteria in the feces of mice with abnormal intestinal flora in a short period of time. It has been found that there is an action to improve abnormal intestinal flora. In addition, although it changed and tested the kind of lactoferrin and the manufacturing method of a drink, the substantially same result was obtained.

Test example 4
This test was conducted in order to examine the combined effect of lactoferrin degradation products.
1) Preparation of sample Prepared a sterilized beverage added with unmodified lactoferrin and lactoferrin degradation product by the same method as in Example 2 except that the lactoferrin content and lactoferrin degradation product content in the beverage were adjusted to 1% respectively. did. A control sample was prepared in the same manner as in Example 2 except that lactoferrin and a lactoferrin degradation product were not added.
2) Test method The test was conducted in the same manner as in Test Example 2 except that the sterilized beverage was raised for 2 weeks.
3) Test results The results of this test are shown in Table 2. As is clear from Table 2, the sterilized beverage to which 1% unmodified lactoferrin and 1% lactoferrin degradation product were added had a remarkable reduction in harmful bacteria in feces as compared to the non-added sterilized beverage. From this result, it was found that the combined use of the lactoferrin degradation product was good.

  In addition, although the same test was done by changing the kind of lactoferrin degradation product, it turned out that there exists the same effect | action which improves an intestinal flora.

[Reference example]
A lactoferrin degradation product was prepared as follows. 50 g of commercially available lactoferrin (manufactured by Olefina of Bergie) was dissolved in purified water at a concentration of 5%, and 1 molar hydrochloric acid was added to adjust the pH to 3. The solution is then heated at 70 ° C. for 20 minutes to hydrolyze, and then adjusted to pH 7 with 1 molar caustic soda solution and lyophilized to obtain 20 g of a lactoferrin degradation product having a degradation rate of 20%. It was.

EXAMPLES Next, although an Example is shown and this invention is demonstrated further in detail, this invention is not limited to a following example.
[Example 1]

  A soft drink raw material liquid containing white grape juice, aloe extract, and lactulose having the following composition was prepared, sterilized at 110 ° C. for 30 seconds, cooled to about 20 ° C., and stored in a sterile tank.

Formulation
Sugar mixed isomerized sugar 11.12 (kg)
White grape juice (6-fold concentrated) 1.67
Aloe extract (5 times concentrated) 1.40
Nonfat dry milk 3.00
Citric acid 0.21
Lactulose 3.00
Fragrance 0.10
Dissolved water 59.50
Separately, 2 kg of bovine lactoferrin (manufactured by Morinaga Milk Industry Co., Ltd., 95% lactoferrin content) is added to 14 kg of room temperature water and dissolved using TK homomixer (manufactured by Tokushu Kika Kogyo Co., Ltd.) to prepare a lactoferrin solution. First lactate (manufactured by Nacalai Tesque, Inc., purity 90%) was added to the lactoferrin solution to adjust the pH to 4.0, and then water was added to adjust the total amount to 20 kg to obtain a 10% lactoferrin solution.

  The lactoferrin solution is sterilized at 155 ° C. for 2 seconds using a steam injection sterilizer (manufactured by Morinaga Milk Industry Co., Ltd.), and the first stage is 20 MPa and the second stage is 2 MPa using a homogenizer (manufactured by Sanmaru Machinery Co., Ltd.). Aseptically homogenized with pressure and mixed with the sterilizing solution stored in the sterilized tank, about 100 kg of a sterilized soft drink containing 2% unmodified bovine lactoferrin was obtained.

  Aseptically sterilized soft drink containing unmodified bovine lactoferrin in aseptic 200ml bottles (made by Tetra Pak Co., Ltd.) 200ml each in a container (Tetra Pak Co.) made of paper, aluminum foil and polyethylene laminate from the outer layer. Thus, 400 sterilized soft drinks containing unmodified bovine lactoferrin were produced.

The resulting sterilized beverage containing native bovine lactoferrin contains 1.9% native bovine lactoferrin, is free from bacterial contamination, and does not generate precipitates even after storage for 6 months at room temperature, maintaining good conditions. Was.
[Example 2]

  A raw material solution of a soft drink containing white grape juice, aloe extract, lactulose and inositol having the following composition was prepared, sterilized at 110 ° C. for 30 seconds, cooled to about 10 ° C., and stored in a sterile tank.

Formulation
Sugar mixed isomerized sugar 11.12 (kg)
White grape juice (6-fold concentrated) 1.67
Aloe extract (5 times concentrated) 1.40
Citric acid 0.21
Lactulose 3.00
Fragrance 0.10
Inositol 0.01
Dissolved water 62.49
Separately, 1 kg of bovine lactoferrin (manufactured by Morinaga Milk Industry Co., Ltd., lactoferrin content 95%) and 1 kg of lactoferrin degradation product prepared by the same method as in the above Reference Example were added to 14 kg of room temperature water, respectively, and TK homomixer (special A lactoferrin solution is prepared using a solution manufactured by Kika Kogyo Co., Ltd., and the pH is adjusted to 4.0 by adding first lactic acid (manufactured by Nacalai Tesque, Inc., purity 90%) to the lactoferrin solution, and then water. Was added to adjust the total amount to 20 kg to obtain a 10% lactoferrin solution.

  The lactoferrin solution is sterilized at 155 ° C. for 2 seconds using a steam injection sterilizer (manufactured by Morinaga Milk Industry Co., Ltd.), and the first stage is 20 MPa and the second stage is 2 MPa using a homogenizer (manufactured by Sanmaru Machinery Co., Ltd.). Aseptically homogenized with pressure. This was added to the sterilizing raw material liquid to prepare about 100 kg of a soft drink containing 1% unmodified bovine lactoferrin and 1% lactoferrin degradation product.

  A soft drink containing 1% unmodified bovine lactoferrin and 1% lactoferrin degradation product is hot-filled into a 500 ml PET resin bottle (manufactured by Yoshino Kogyo), cooled to 40 ° C. or lower, and boiled soft drink with unmodified bovine lactoferrin 200. Pieces were manufactured.

The resulting sterilized beverage containing native bovine lactoferrin contains 0.8% of native bovine lactoferrin and 0.9% of native lactoferrin degradation product, without bacterial contamination, and after storage for 6 months at room temperature. Also, no precipitate was formed, and a good state was maintained.
[Reference example]

1) Preparation of raw material liquid Raw milk was passed through a disk-type cream separator to prepare skim milk and cream.
2) Separation of lactoferrin CM-Toyopar 650 (manufactured by Toyo Soda) 500 ml having a carboxymethyl group as an ion exchange group, a hemoglobin adsorption capacity of 4.6 g / 100 ml, and a volume change of 1.0 Was packed into a column having an inner diameter of 10 cm, and 10% saline solution was passed through, followed by washing with water to prepare an Na-type ion exchanger.

  Subsequently, the skim milk was passed through the ion exchanger at a temperature of 4 ° C. and a flow rate of 4 l / h. The skim milk after passing showed no change in appearance and flavor.

After passing through the skimmed milk, water was passed through the column and washed, and 10% saline was passed at a flow rate of 5 l / h to desorb the ion exchanger adsorbing component to obtain 5.0 l of a recovered solution. During this time, no change in head volume was observed. The lactoferrin concentration in this recovered solution was 36 mg / 100 ml.
3) Heat sterilization of raw material liquid The cream obtained in 1) above and the skim milk after passing through the column in 2) were mixed and stirred in a cold storage tank, and then a steam injection sterilizer ( Sterilized at 155 ° C. for 2 seconds by Morinaga Milk Industry Co., Ltd., and aseptically homogenized at a pressure of 30 MPa in the first stage and 3 MPa in the second stage using a homogenizer (manufactured by Sanmaru Kikai Kogyo Co., Ltd.) and heated Sterilized milk was obtained and stored in a sterile tank.
4) Heat sterilization of lactoferrin and preparation of sterilized beverage First lactate (manufactured by Nacalai Tesque, Inc., purity 90%) was added to the lactoferrin-containing recovered liquid obtained in 2) above to adjust the pH to 4.0. -Sterilized at 155 ° C for 2 seconds using a mu-injection type sterilizer (manufactured by Morinaga Milk Industry Co., Ltd) And mixed with the heat-sterilized milk stored in the sterilized tank in 3) above.

  Through the above steps, heat-sterilized milk having almost the same components as raw milk was obtained, but the degree of denaturation of lactoferrin in this heat-sterilized milk was determined using a reverse phase column (Asahi Pack C4P-50: trademark, As a result of testing by high performance liquid chromatography using gradient elution of acetonitrile and 0.5 molar sodium chloride using Asahi Kasei Co., Ltd., it was confirmed that the denaturation rate was 0% and lactoferrin was not denatured at all.

[Comparative example]
The same raw milk as in Example 3 was heat sterilized by the same apparatus and conditions as in 3) in the conventional method, ie, Example 3 to obtain heat sterilized milk.
The degree of denaturation of lactoferrin in the heat pasteurized milk obtained was tested by the same method as in Example 3. As a result, the denaturation rate was 100%, and it was confirmed that lactoferrin was completely inactivated.

As described above in detail, the present invention is a sterilized beverage containing unmodified lactoferrin, which is heat-sterilized and mixed with unmodified lactoferrin in a sterilized beverage material solution (excluding soymilk material solution), and a method for producing the same The effects obtained by the present invention are as follows.
1) Improve the bacterial composition of human or animal abnormal intestinal flora by synergistic effects of native lactoferrin and lactulose bifidobacterial growth factor.
2) Since the soft drink containing unmodified lactoferrin of the present invention having the above-mentioned effects is heat-sterilized, it can be stored for a long period of time and can be immediately drunk from a container.
3) The sterilized beverage containing unmodified lactoferrin of the present invention does not deteriorate in quality due to storage.

Claims (7)

A sterilized beverage containing unmodified lactoferrin mixed with heat-sterilized and unmodified lactoferrin in a sterilized beverage material solution (excluding soymilk material solution) containing aloe extract and lactulose .       The sterilized beverage containing unmodified lactoferrin according to claim 1, which is heat-sterilized and contains 0.02-5.0% (mass) of unmodified lactoferrin.       The sterilized beverage containing unmodified lactoferrin according to claim 1 or 2, wherein the heat-sterilized lactoferrin remains active at a rate of 80 to 100% (mass). The following steps f) to h);
f) A step of sterilizing and cooling a beverage raw material liquid (excluding soy milk raw material solution) containing aloe extract and lactulose ,
g) A step of adding an acid to the lactoferrin solution, adjusting the solution to acidity, and heat-sterilizing; and h) heat-sterilized unmodified lactoferrin in the sterilized beverage raw material solution by 0.02 to 5. Adding at a rate of 0% (mass);
A method for producing a sterilized beverage containing unmodified lactoferrin, comprising:   The method for producing a sterilized beverage containing unmodified lactoferrin according to claim 4, wherein the heat-sterilized lactoferrin remains active at a rate of 80 to 100% (mass).   The method for producing a sterilized beverage containing unmodified lactoferrin according to claim 4 or 5, wherein the heat sterilization of the lactoferrin solution is performed by heating at a temperature of 150 to 155 ° C for 2 to 5 seconds.   The method for producing an unmodified lactoferrin-containing sterilized beverage according to any one of claims 4 to 6, wherein the lactoferrin solution is adjusted to an acidity in the range of pH 2.0 to 5.5.