JP4764380B2 - Fermented milk - Google Patents

Description

The present invention relates to a fermented milk having a good appearance without separation or precipitation of milk components even though hyaluronic acid and / or a salt thereof is blended.

Hyaluronic acid is a mucopolysaccharide present in living organisms, particularly subcutaneous tissues, and has been widely used as a raw material for cosmetics as hyaluronic acid or a salt thereof due to its high moisturizing function. In addition, by taking orally hyaluronic acid or a salt thereof, an effect of supplementing a decrease in hyaluronic acid content inherent in the living body and improving skin moisture retention, elasticity, and flexibility has been recognized.

In recent years, since interest in functional foods has been improved, many foods containing hyaluronic acid and its salts have been developed. Among them, synergistic effects on health and beauty can be expected by adding hyaluronic acid to fermented milk.

For this reason, development about fermented milk which mix | blended hyaluronic acid and its salt is made | formed, for example, by adding hyaluronic acid to a dairy product in Unexamined-Japanese-Patent No. 2006-522591 (patent document 1), It is described that it can affect the texture properties of the product, in particular the viscosity, and increase the gel rigidity. Japanese Patent Laid-Open No. 10-56983 (Patent Document 2) describes a method of adding hyaluronic acid to a gelled food, and an example of yogurt is given as the gelled food. Therefore, the inventor added hyaluronic acid (trade name “Hyaluron San HA-F”, manufactured by QP Corporation, average molecular weight of 800,000) used in Examples of Patent Document 1 to milk to produce yogurt. Milk was separated and precipitation occurred, and it was difficult to say that the appearance was satisfactory.

JP 2006-522591 A Japanese Patent Laid-Open No. 10-56983

Accordingly, an object of the present invention is to provide fermented milk having a good appearance without separation or precipitation of milk components despite the blending of hyaluronic acid and / or a salt thereof.

As a result of intensive studies on hyaluronic acid to achieve the above object, the present inventor unexpectedly separated milk components if hyaluronic acid and / or a salt thereof having an average molecular weight below a specific value was blended. The inventors found that fermented milk having a good appearance and no precipitation can be obtained, and the present invention has been completed.

That is, the present invention
(1) Fermented milk comprising hyaluronic acid having an average molecular weight of 300,000 or less and / or a salt thereof,
(2) fermented milk according to (1), comprising hyaluronic acid and / or a salt thereof having an average molecular weight of 50,000 or less,
(3) Fermented milk according to any one of (1) and (2), comprising hyaluronic acid having an average molecular weight of 20,000 or less and / or a salt thereof,
(4) The molecular weight distribution of hyaluronic acid and / or a salt thereof is such that the proportion of components having a molecular weight of 10,000 or less is 40% or more and the proportion of components having a molecular weight of 50,000 or more is 5% or less (2) or (3) Any fermented milk,
(5) The fermented milk according to any one of (1) to (4), wherein the blending amount of hyaluronic acid and / or a salt thereof is 0.001 to 10%,
It is.

ADVANTAGE OF THE INVENTION According to this invention, although hyaluronic acid and / or its salt are mix | blended, fermented milk without isolation | separation and precipitation of a milk component can be provided. Therefore, further expansion of utilization of hyaluronic acid and / or a salt thereof in the food market can be expected.

Hereinafter, the present invention will be described in detail. In the present invention, “%” means “mass%”.

The fermented milk of the present invention is obtained by fermenting milk or milk containing non-fat milk solid content equal to or higher than this with lactic acid bacteria or yeast to make paste or liquid, or by freezing it. Type yogurt, hard type fruit yogurt, drinking yogurt, frozen yogurt and the like.

The present invention is characterized in that the fermented milk contains hyaluronic acid having an average molecular weight of 300,000 or less and / or a salt thereof. Here, “hyaluronic acid” refers to a polysaccharide having one or more repeating structural units composed of disaccharides of glucuronic acid and N-acetylglucosamine. The “hyaluronic acid salt” is not particularly limited, but is preferably a food or pharmaceutically acceptable salt, for example, sodium salt, potassium salt, calcium salt, zinc salt, magnesium salt, ammonium salt, etc. Is mentioned.

The hyaluronic acid or a salt thereof used in the present invention has an average molecular weight of 300,000 or less, preferably 50,000 or less, more preferably 20,000 or less. When the average molecular weight of hyaluronic acid or a salt thereof is larger than the above value, when the hyaluronic acid and / or a salt thereof is blended into a dairy product, separation or precipitation of milk components occurs, and the desired appearance of the present invention is good. This is because it does not become a fermented milk. In the present invention, the lower limit of the average molecular weight is not limited, but an average molecular weight of 5,000 or more is preferable in consideration of industrial productivity of hyaluronic acid or a salt thereof having a high degree of purification.

The measurement method of the average molecular weight prescribed | regulated by this invention is demonstrated.

（式２）

The average molecular weight defined in the present invention is a molecular weight calculated from the intrinsic viscosity of hyaluronic acid and / or a salt thereof. In order to determine the intrinsic viscosity of hyaluronic acid and / or a salt thereof, first, an aqueous solution of a plurality of hyaluronic acid and / or a salt thereof is prepared, and hyaluronic acid and / or the solution thereof in an Ubbelohde viscometer (Shibata Scientific Instruments Co., Ltd.) is prepared. The specific viscosity and the reduced viscosity are calculated based on the following formulas 1 and 2 from the flowing seconds of the aqueous salt solution and the flowing seconds of the solvent. At this time, an Ubbelohde viscometer having a coefficient such that the number of seconds of flow is 200 to 1000 seconds is used. The concentration of the aqueous solution of hyaluronic acid and / or a salt thereof is selected to be suitable for the measuring instrument. The measurement is performed in a constant temperature water bath at 30 ° C. so that there is no temperature change.
(Formula 1)

(Formula 2)

Next, for each aqueous solution of hyaluronic acid and / or salt thereof, a calibration curve was prepared by plotting the obtained reduced viscosity on the vertical axis and the hyaluronic acid and / or salt concentration in terms of dry matter on the horizontal axis, By extrapolating hyaluronic acid and / or its salt concentration to zero, the intrinsic viscosity of hyaluronic acid and / or its salt is obtained. Based on the following formula 3, the average molecular weight M can be determined from the intrinsic viscosity of hyaluronic acid and / or a salt thereof.
(Formula 3)
Intrinsic viscosity (dL / g) = K′M ^α
(In the above formula 3, K ′ = 0.036 and α = 0.78.)

The hyaluronic acid and / or salt thereof used in the present invention has an average molecular weight of 300,000 or less, preferably 50,000 or less, and more preferably 20,000 or less. It is preferable that the ratio is 40% or more and the ratio of components having a molecular weight of 50,000 or more is 5% or less. Use of hyaluronic acid and / or a salt thereof having such a molecular weight distribution is effective in that separation and precipitation of milk components hardly occur even when blended in dairy products at a high concentration.

The molecular weight distribution defined in the present invention can be obtained by liquid chromatography analysis of hyaluronic acid and / or a salt thereof using a gel filtration column. Hyaluronic acid and / or a salt thereof is a mixture of a plurality of components having different molecular weights depending on the number of repeating structural units (N-acetyl-D-glucosamine and D-glucuronic acid). Accordingly, by performing liquid chromatography analysis on an aqueous solution of hyaluronic acid and / or a salt thereof using a gel filtration column, the components constituting hyaluronic acid and / or the salt thereof can be separated by molecular size.

The molecular weight distribution of hyaluronic acid and / or a salt thereof in the present invention is determined by HPLC analysis device (trade name “Alliance PDA System”, manufactured by Nippon Waters Co., Ltd.) and gel filtration column (trade name “Diol-120”, manufactured by YMC Co., Ltd.). ) And a 0.1% (w / v) aqueous solution of hyaluronic acid and / or its salt as an analytical sample, and this analytical sample can be measured by liquid chromatography analysis.

The conditions for liquid chromatography analysis are as follows.
Column temperature: 40 ° C
Flow rate: 1 mL / min Injection volume of 0.1% (w / v) aqueous solution of hyaluronic acid and / or its salt: 20 μL
Mobile phase: 0.003 mol / L phosphate buffer (containing 0.15 mol / L NaCl, pH 7.0)
Ultraviolet detector: measured at λ = 210 nm In the liquid chromatography using the gel filtration column according to the present invention, the longer the retention time, the lower the molecular weight. In order of decreasing retention time, N-acetylglucosamine, D-glucuronic acid, hyaluronic acid (disaccharide: one repeating structural unit), hyaluronic acid (tetrasaccharide: two repeating structural units), hyaluronic acid (hexasaccharide: repeating structure) Peaks of 3 units), hyaluronic acid (octasaccharide: 4 repeating structural units)... Are obtained. The retention time and molecular weight at each peak are calculated, and a calibration curve of this retention time versus molecular weight is obtained (Formula 4).

In formula 4 described later, x represents a retention time, and y represents a molecular weight. Next, from the calibration curve shown in Equation 4, a retention time corresponding to a predetermined molecular weight (10,000 or 50,000) is calculated, and by dividing the peak by these retention times, the components in the predetermined molecular weight range are calculated. Find the percentage. The molecular weight indicated by each peak is referred to the peak in the chromatogram obtained by liquid chromatography analysis of the smallest structural unit (disaccharide) of hyaluronic acid and / or its salt with a known molecular weight in the same manner. To be identified.

For example, the ratio of components having a molecular weight of 10,000 or less is calculated by calculating a retention time corresponding to a molecular weight of 10,000 from the calibration curve shown in Equation 4, and dividing the absorption area of the component after this retention time by the total absorption area. Can be sought. Similarly, for the proportion of components having a molecular weight of 50,000 or more, the retention time corresponding to the molecular weight of 50,000 is calculated from the calibration curve shown in Equation 4, and the absorption area of the component is divided by the total absorption area before this retention time. It can ask for.

As an example, Table 1 shows the relationship between the number of repeating units of each molecular weight component obtained from the chromatogram shown in FIG.

(Formula 4)
y = -21.4x < ³ > + 1296.2x < ² > -267477.1x + 189427.1

A typical method for producing low molecular weight hyaluronic acid and / or a salt thereof having an average molecular weight of 300,000 or less, preferably 50,000 or less, more preferably 20,000 or less used in the present invention will be described below. In addition, the manufacturing method of the low molecular hyaluronic acid and / or its salt used by this invention is not limited to this.

Hyaluronic acid and / or a salt thereof (hereinafter also referred to as “raw hyaluronic acid and a salt thereof”), which is a raw material for the low molecular hyaluronic acid and / or salt thereof, is generally a living organism such as a chicken crown, umbilical cord, eyeball, skin, cartilage, and the like. It is obtained by extracting (and purifying as necessary) these raw materials from a culture solution obtained by culturing hyaluronic acid-producing microorganisms such as tissues or microorganisms belonging to the genus Streptococcus. For example, the molecular weight of the raw material hyaluronic acid and / or its salt extracted from a chicken crown is usually 2 million to 8 million.

The low molecular weight hyaluronic acid and / or salt thereof is produced by dispersing the raw material hyaluronic acid and / or salt thereof in an acidic water-containing medium and removing the acidic water-containing medium by heating and drying. be able to. Here, the degree of low molecular weight can be adjusted by adjusting the stirring speed and stirring time. Moreover, by performing the above-mentioned dispersion | distribution process on 30-70 degreeC on the heating conditions for less than 1 hour, it can be made low molecular weight stably to the target molecular weight. More specifically, the dispersion medium obtained by adding the powdered raw material hyaluronic acid and / or salt thereof to the acidic water-containing medium while stirring can be heated. Alternatively, the acidic water-containing medium may be heated in advance, and raw material hyaluronic acid and / or a salt thereof may be added thereto to maintain the temperature.

In the above production method, the water-containing medium refers to a dispersion medium of hyaluronic acid and / or a salt thereof containing water. The medium that can be used as the water-containing medium preferably has low solubility of hyaluronic acid and / or a salt thereof. The medium that can be used as the water-containing medium is not particularly limited, but for example, a liquid that has a property of being dissolved in water and that can be used in the production process of food or medicine is preferable. Examples of the medium that can be used as the water-containing medium include alcohol-based media (for example, methanol, ethanol, n-propanol, 2-propanol, etc.), ketone-based media (for example, acetone, methyl ethyl ketone, etc.), tetrahydrofuran, acetonitrile, and the like. These can be used alone or in combination. Among these, it is preferable that it is at least 1 sort (s) chosen from methanol, ethanol, and acetone at the point of the low boiling point and a price. The water content in the water-containing medium is not particularly specified, but if the water content is large, the hyaluronic acid and / or salt thereof cannot be maintained in a dispersed state and is dissolved in the water-containing medium, which may lead to a decrease in yield. Therefore, the ratio of water to the total amount of the water-containing medium is preferably 40% capacity or less, more preferably 30% capacity or less.

Moreover, in the said manufacturing method, as what is used in order to make a water-containing medium acidic, acids, such as hydrochloric acid and a sulfuric acid, and acidic cation exchange resin are mentioned, for example.

A method of converting a low molecular weight hyaluronic acid having an average molecular weight of 300,000 or less, preferably 50,000 or less, more preferably 20,000 or less into a hyaluronic acid salt, and conversion from the low molecular hyaluronic acid salt to hyaluronic acid The method to do is not specifically limited. As a method for converting the low molecular weight hyaluronic acid into a hyaluronic acid salt, for example, an alkaline aqueous solution (for example, an aqueous solution of sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, ammonium hydroxide, etc.) is used. And processing method. Examples of the method for converting the low molecular weight hyaluronic acid salt into hyaluronic acid include, for example, a method of treating with an acid aqueous solution (for example, an aqueous solution of hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, etc.), an acidic cation, and the like. A method using an exchange resin may be mentioned.

In the fermented milk of the present invention, the blending amount of low molecular weight hyaluronic acid and / or a salt thereof having an average molecular weight of 300,000 or less, preferably 50,000 or less, more preferably 20,000 or less obtained by the above-described production method is 0. About 0.001 to 10% is preferable, and about 0.005 to 2% is more preferable. If the blending amount of hyaluronic acid and / or salt thereof is more than the above range, the flavor of hyaluronic acid and / or salt thereof becomes strong and the original taste of fermented milk is impaired. On the other hand, if the amount is less than the above range, the cosmetic effect of hyaluronic acid and / or a salt thereof hardly appears and the commercial value is lowered, which is not preferable.

The method for producing fermented milk of the present invention comprises the above-described low molecular weight hyaluronic acid or a salt thereof having an average molecular weight of 300,000 or less, preferably 50,000 or less, more preferably 20,000 or less, which is the essential ingredient of the present invention. Or milk containing a non-fat milk solid content equal to or higher than this, or a method including a step of blending with fermented milk, is not particularly limited. As a general method for producing fermented milk, fermented milk ingredients that have been fermented and cooled in a tank are pre-fermented and crushed and filled into individual containers for distribution, and a certain amount of starter is added. After filling the mix into a single-use container for distribution, the mixture is roughly classified into a post-fermentation type that is fermented until reaching a predetermined degree of lactic acid in the fermentation chamber and then cooled. The present invention may be any method.

This invention can mix | blend suitably the raw material generally used for fermented milk in the range which does not impair the effect of this invention. Examples of such raw materials include sugar, powdered sugar, granulated sugar, fructose, starch syrup, honey, fructose, palatinose, trehalose, lactose, xylose and other sugars, agar, gelatin, starch, carrageenan, xanthan gum, dextrin, pectin, etc. Texture improver, sugar alcohols such as sorbitol, maltitol, xylitol, erythritol, reduced starch syrup, reduced maltose starch syrup, high sweetness sweeteners such as sucralose, stevia, aspartame, acid materials such as citric acid, lactic acid, vitamins Examples include vitamins such as A, vitamin B, vitamin C, and vitamin D, cosmetic raw materials such as collagen and water-soluble dietary fiber, dairy products, milk protein, pigments, fruit juice, vegetable oils, egg yolk, and fragrances. In addition, dispersible solid foods such as strawberry, blueberry, mandarin orange, pineapple, kiwi, melon, peach, apple and other pulps, jams, and nata de coco can be included.

Hereinafter, fermented milk using low molecular weight hyaluronic acid and / or a salt thereof having an average molecular weight of 300,000 or less, preferably 50,000 or less, and more preferably 20,000 or less used in the present invention will be specifically described based on Examples and the like. To do. The present invention is not limited to these.

[Preparation Example 1] Hyaluronic acid with an average molecular weight of 9000 In this preparation example, a sodium hyaluronate (hereinafter also referred to as “HANa”) fine powder extracted and purified from a chicken crown was prepared as a raw material. The raw material HANa had an average molecular weight of about 2.1 million and a purity of 97%.

First, a 300 L tank equipped with a stirrer and a jacket was filled with 110 L of 0.5% sulfuric acid-containing 80% water-containing acetone (acidic water-containing medium), and heated with stirring to a liquid temperature of 60 ° C. Here, 80% water-containing acetone contains 80 (w / w)% acetone and 20 (w / w)% water, and 80% acetone containing 0.5% sulfuric acid contains 0% sulfuric acid. 0.5 (w / w)% and 80% water-containing acetone is contained 99.5 (w / w)%. After reaching 60 ° C., 6 kg of the prepared raw material HANa fine powder was charged into the tank while stirring. While heating so as to maintain the temperature of the sulfuric acid-containing water-containing acetone at 60 ° C., the raw material HANa fine powder was stirred so as to be in a dispersed state.

Next, after stirring for 15 minutes and allowing to stand, the supernatant sulfuric acid-containing water-containing acetone was removed by decantation to obtain a precipitate. To the obtained precipitate, 110 L of 80% water-containing acetone containing 0.5% sulfuric acid previously heated to 60 ° C. was added, and similarly stirred for 15 minutes while heating to 60 ° C. This operation was repeated a total of 3 times.

Next, 110 L of 80% water-containing acetone was added to the precipitate obtained after removing sulfuric acid-containing water-containing acetone, and stirring was performed for 15 minutes for the purpose of removing sulfuric acid. This operation was repeated until no sulfuric acid remained.

Furthermore, water-containing acetone was removed by decantation to obtain a residue. The residue was further centrifuged to remove water-containing acetone, and then dried by heating at 70 ° C. under reduced pressure for 12 hours using a vacuum dryer.

Through the above steps, 5.3 kg of hyaluronic acid (yield: about 88%) was obtained as white fine powder. This hyaluronic acid has an average molecular weight of 9,000 converted from intrinsic viscosity, and in the molecular weight distribution, the proportion of components having a molecular weight of 10,000 or less is 49% or more and the proportion of components having a molecular weight of 50,000 or more is 0.5%. there were.

[Preparation Example 2] Sodium hyaluronate having an average molecular weight of 35,000 In this preparation example, the HANa fine powder used in Preparation Example 1 was prepared as a raw material.

First, a 300 L tank equipped with a stirrer was filled with 110 L of 73% water-containing ethanol (acidic water-containing medium) containing 2% hydrochloric acid, and heated to 50 ° C. while stirring. Here, 73% hydrous ethanol contains 73 (w / w)% ethanol and 27 (w / w)% water, and 73% hydrous ethanol containing 2% hydrochloric acid contains 2 ( w / w)%, and it contains 98% (w / w)% of 73% water-containing ethanol. After the temperature reached 50 ° C., 6 kg of the prepared raw material HANa fine powder was charged into the tank while stirring. While heating to maintain the temperature of the hydrochloric acid-containing aqueous ethanol at 50 ° C., the raw material HA powder was stirred so as to be in a dispersed state.

Next, after stirring for 15 minutes and allowing to stand, the supernatant hydrochloric acid-containing aqueous ethanol was removed by decantation to obtain a precipitate. To the obtained precipitate, 110 L of 73% water-containing ethanol containing 2% hydrochloric acid previously heated to 50 ° C. was added, and similarly stirred for 15 minutes while heating to 50 ° C. This operation was repeated a total of 3 times.

Next, 110 L of 73% aqueous ethanol was added to the precipitate obtained after removing the hydrochloric acid-containing aqueous ethanol, and the mixture was stirred for 15 minutes for the purpose of removing hydrochloric acid. This operation was repeated until no hydrochloric acid remained.

Furthermore, the water-containing ethanol was removed by decantation to obtain a residue. The residue was further centrifuged to remove water-containing ethanol and then dissolved again in 100 L of water in the same tank to prepare an aqueous solution. While stirring the aqueous solution, a 20% sodium hydroxide solution was added to the aqueous solution to adjust the pH to 6.5. Subsequently, this aqueous solution was spray-dried with a spray dryer.

By the following steps, 5.1 kg (yield of about 85%) of low molecular weight sodium hyaluronate as a white fine powder was obtained. This hyaluronic acid has an average molecular weight converted to intrinsic viscosity of 35,000, and in the molecular weight distribution, the proportion of components having a molecular weight of 10,000 or less is less than 40% and the proportion of components having a molecular weight of 50,000 or more is more than 1%. Met.

[Example 1]
Fermented milk having the following composition was produced. That is, hyaluronic acid having an average molecular weight of 9,000 obtained in Preparation Example 1 was dissolved in fresh water and mixed with milk and skim milk powder. Next, after heat sterilization treatment at 90 ° C. for 10 minutes, the mixture was cooled to 40 ° C., a yogurt starter was added, and the mixture was stirred uniformly, and dispensed in 80 mL portions into 100 mL individual food containers. This was fermented at 40 ° C., and when the lactic acid acidity reached around 0.7%, it was cooled to 10 ° C. or less, and the fermentation was stopped to produce fermented milk.

<Combination ratio>
80% milk
Nonfat dry milk 3%
Yogurt starter (commercial yogurt) 3%
Hyaluronic acid (Preparation Example 1) 0.0125% / 0.075% / 0.15%
Shimizu Residual -――――――――――――――――――――――――――――――――――――――
100%

[Example 2]
Fermented milk was produced in the same manner as in Example 1 except that sodium hyaluronate having an average molecular weight of 35,000 obtained in Preparation Example 2 was used in place of the hyaluronic acid used in the fermented milk of Example 1. .

Example 3
Instead of the hyaluronic acid used in the fermented milk of Example 1, a hyaluronic acid having an average molecular weight of 300,000 (trade name “Hyaluronic Sun HA-LF”, manufactured by QP Corporation) was used in the same manner as in Example 1. Fermented milk was made.

[Comparative Example 1]
Fermented milk was produced in the same manner as in Example 1 except that sodium hyaluronate having an average molecular weight of 800,000 was used instead of hyaluronic acid used in the fermented milk of Example 1.

[Comparative Example 2]
Instead of the hyaluronic acid used in the fermented milk of Example 1, an hyaluronic acid having an average molecular weight of 1.3 million (trade name “Hyaluron San HA-LQ”, manufactured by QP Corporation) was used in the same manner as in Example 1. Fermented milk was made.

[Test Example 1]
The fermented milk obtained in Examples 1 to 3 and Comparative Examples 1 to 2 was taken out of the container, and the state of the contents was visually evaluated based on the control product shown below. The evaluation results are shown in Table 2.

[Control product]
In the fermented milk of Example 1, fermented milk was produced in the same manner except that the hyaluronic acid of Preparation Example 1 was not added.

From Table 2, the fermented milk of Examples 1 to 3 blended with hyaluronic acid and / or a salt thereof having an average molecular weight of 300,000 or less according to the present invention blended hyaluronic acid and / or a salt thereof with an average molecular weight exceeding 300,000. There was no separation of milk confirmed in Comparative Example 1 or 2, and the properties were good. In addition, the fermented milk of Example 1 or 2 blended with hyaluronic acid having an average molecular weight of 50,000 or less and / or a salt thereof had almost the same appearance as the control product. In particular, blending with hyaluronic acid having an average molecular weight of 20,000 or less, a molecular weight distribution in which the proportion of components with a molecular weight of 10,000 or less is 40% or more, and the proportion of components with a molecular weight of 50,000 or more is 5% or less The fermented milk of Example 1 had the same properties as the control product even when the amount of hyaluronic acid was large.

FIG. 1 shows an example of a chromatogram of hyaluronic acid.

Claims (1)

Fermented milk, characterized by containing an average molecular weight 35,000 or less of hyaluronic acid and / or a salt thereof from 0.0125 to 0.15%.

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