JP3522373B2 - Lactic acid bacteria growth promoter - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a substance which promotes the growth of bacteria belonging to the genera Bifidobacterium and Lactobacillus, which are enterobacteria beneficial to humans, and in which beneficial bacteria form a predominant intestinal flora. .

[0002]

2. Description of the Related Art Many bacteria are resident in the human intestine,
It forms a complex intestinal flora.

Of the bacteria that make up this intestinal flora,
It is known that lactic acid bacteria such as Bifidobacterium and Lactobacillus have beneficial effects on humans such as synthesis of vitamins, protection against infection by foreign pathogens, and enhancement of immune function.

On the other hand, Clostridium perfringens and the like produce putrefaction products such as ammonia and indole and bacterial toxins, and cause colon cancer, arteriosclerosis, hypertension, liver damage, aging and the like. In order for humans to maintain their health, it is important to maintain the intestinal flora in a state in which beneficial bacteria represented by Bifidobacterium are dominant.

From such a point of view, many substances have been found so far that proliferate Bifidobacterium, which is a typical intestinal beneficial bacterium. For example, dried potatoes (Japanese Patent Application Laid-Open No. H6-6
No. 217733), a leaf extract of coffee genus plant (JP-A-6-125771), a syrupy substance extracted from fruit of evening glory (JP-A-2-13588), and a solvent extract of Araliaceae plant ( JP-A-2-2494
No. 82), an aqueous alcoholic solution extract of a fat-soluble component extraction residue of soybean (JP-A-62-155082, JP-A-60-66978), and an enzyme-decomposed product of a barley protein-containing substance (JP-A-62). 61-282070) and the like.

[0006] However, these substances have problems such as insufficient effect and promotion of growth of unwanted bacteria other than lactic acid bacteria.

[0007]

Therefore, an object of the present invention is to provide a substance which selectively promotes growth of lactic acid bacteria and forms beneficial intestinal flora in which intestinal flora is predominant.

[0008]

As a result of various studies, the inventors of the present invention have found that cacao beans, particularly cacao husks, which are their outer coats, contain substances that grow lactic acid bacteria belonging to the genera Bifidobacterium and Lactobacillus. The present invention was completed by succeeding in extracting the substance as a growth promoter showing a selective effect on lactic acid bacteria. Up to now, it has not been known at all that cocoa beans contain a substance that promotes the growth of lactic acid bacteria, and it was first discovered by the present inventors.

That is, the present invention is a lactic acid bacterium growth-promoting substance containing, as an active ingredient, an extraction residue of an organic solvent which is compatible with water of cocoa beans and / or cocoa husk or a water extract of the residue.

Although cocoa beans can be used in their entirety, in the practice of the present invention, it is desirable to use cacao husk, which is the outer coat of cocoa beans, from the viewpoint of effective use, although it was conventionally treated as an unnecessary substance. . These cocoa beans and cocoa husks can be heated as well as raw ones.

A residue obtained after extracting the cocoa beans and / or cocoa husk with an organic solvent compatible with water or the residue is extracted with water, and then water is distilled off to extract water, which is the object of the present invention. Get things.

Solvents compatible with water used for extraction include organic solvents such as methanol, ethanol, propanol, butanol and acetone, derivatives thereof such as ethyl acetate and methyl acetate, compounds of two or more kinds of these, for example. Examples include polar solvents such as acetone alcohol and mixtures of two or more thereof. Non-polar organic solvents such as hexane and ether and water hardly give the lactic acid bacterium growth promoting substance which is the object of the present invention.

The object of the present invention is that most of the residue of extraction with an organic solvent compatible with water is contained in the extraction product obtained by extracting cocoa beans and / or cocoa husk with an organic solvent. The product has a strong odor characteristic of cocoa beans, but almost no lactic acid bacterium growth promoting effect is observed.

The water extract obtained by extracting the residue after extraction with an organic solvent with water shows a strong growth promoting effect on lactic acid bacteria, but has almost no growth promoting effect on other intestinal bacteria. It has properties not shown. In addition, since the aroma substances contained in cacao beans and cacao husks are extracted with an organic solvent, the obtained water extract is brown and odorless fine powder.

Further, in the present invention, a fraction having a molecular weight of 10,000 or less may be collected from the water extract obtained as described above by using a separating means such as an ultrafiltration membrane and used as an active ingredient. That is, the molecular weight of the water extract is 10,000.
Although the following fractions have a strong lactic acid bacterium growth promoting effect, it has been confirmed that the fractions having a molecular weight of more than 10,000 are very weak.

An example of the present invention will be described below.

[0017]

Example 1 2400 g of ethanol was added to 1000 g of cacao husk heated at 160 ° C. for 1 hour, and reflux extraction was performed at 78 ° C. for 1 hour. The liquid thus treated was separated into an extract and a residue by centrifugation, and 6100 g of water was added to the obtained residue,
A water extract was prepared by heating at 40 ° C. for 30 minutes for extraction. Water was distilled off from the obtained water extract to obtain 100 g of a brown, odorless water extract fine powder. The powder was dissolved in 10 times the amount of water and the pH was measured. As a result, the pH was 5.2.

Example 2 In the same manner as in Example 1 except that acetone was used in place of ethanol, brown and odorless water extract fine powder 112 g was prepared. The powder was dissolved in 10 times the amount of water and the pH was measured. As a result, the pH was 5.2.

Example 3 107 g of brown and odorless water extract fine powder were obtained in the same manner as in Example 1 except that raw cocoa husk which was not heated was used.
Was prepared. The powder was dissolved in 10 times the amount of water and the pH was measured. As a result, the pH was 5.1.

Example 4 A brown, odorless water extract fine powder 133 g was prepared in the same manner as in Example 1 except that 1000 g of cocoa beans heated at 160 ° C. for 1 hour was used. The powder was dissolved in 10 times the amount of water and the pH was measured. As a result, the pH was 5.1.

Example 5 A brown, odorless water extract fine powder 125 g was prepared in the same manner as in Example 1 except that 1000 g of unheated raw cocoa beans was used. The powder was dissolved in 10 times the amount of water and the pH was measured. As a result, the pH was 5.2.

Comparative Example 1 In Example 1, ethanol was distilled off from the extract obtained by centrifugation to obtain 48 g of an ethanol extract fine powder which was brown and had a strong odor characteristic of cocoa beans. The powder was dissolved in 10 times the amount of water and the pH was measured. As a result, the pH was 3.7.

Comparative Example 2 6100 g of water was added to 1000 g of cacao husk heated at 160 ° C. for 1 hour, and the mixture was heated and extracted at 40 ° C. for 30 minutes to prepare a water extract. Water was distilled off from the obtained water extract to obtain 120 g of a brown water extract fine powder having a strong odor characteristic of cocoa beans. Dissolve this powder in 10 times the amount of water, p
As a result of measuring H, the pH was 4.7.

Comparative Example 3 To 1000 g of cacao husk heated at 160 ° C. for 1 hour, 2400 g of hexane was added, and after reflux extraction at 60 ° C. for 1 hour, 6100 g of water was added to the hexane extraction residue and heated at 40 ° C. for 30 minutes for extraction. To prepare a water extract. Water was distilled off from the resulting water extract to obtain 127 g of brown and odorless water extract fine powder. Dissolve this powder in 10 times the amount of water, p
As a result of measuring H, the pH was 4.7.

Test Example 1 The effect of six samples of Examples 1 to 3 and Comparative Examples 1 to 3 on the growth of lactic acid bacteria and other intestinal bacteria was examined.

The test strain was applied to Brix River Broth 37
After preculture at 48 ° C. for 48 hours, the bacterial cells were washed with physiological saline and suspended in 6 ml of physiological saline. 20 μl of this bacterial suspension
Was added so that the concentration of the test substance would be 0.6% 6
After inoculating ml of the test medium having the composition shown in Table 1 and anaerobically culturing at 37 ° C. for 48 hours, the pH of the culture solution was measured to examine the growth of the bacteria. The growth-promoting effect is "weakly effective" when the pH is lowered by 0.1 to 0.5 as compared with the control, by inoculating the medium in which the test substance is not added with the bacteria and similarly culturing; (+ ), When there was a decrease of 0.6 to 1.0, "effective"; +, when there was a decrease of 1.1 to 1.5, "strong effect";

[0027]

[Table 1]

The results are shown in Table 2. The water extracts of Examples 1 to 3 promoted the growth of many bacteria belonging to the genus Bifidobacterium and Lactobacillus, but not to other enterobacteria. Did not promote growth except for one strain of Eubacterium.

[0029]

[Table 2]

From this, it was shown that the water extract of Example 1 has an effect of selectively growing lactic acid bacteria belonging to the genus Bifidobacterium and Lactobacillus, which are useful bacteria among the enterobacteria. .

On the other hand, the ethanol extract of Comparative Example 1 and the water extracts of Comparative Examples 2 and 3 showed almost no effect of promoting the growth of lactic acid bacteria. As a cause of this, cacao husk contains growth-promoting substances in addition to growth-promoting substances for lactic acid bacteria, and it is considered that these substances may be removed by extraction with an organic solvent compatible with water.

Test Example 2 The following test was conducted to examine the stability of the lactic acid bacterium growth promoting substance of the present invention against acid and digestive enzymes. The outline of the test is shown in FIG.

Concentrated hydrochloric acid was added to the 50% (w / w) aqueous solution of the water extract of Example 1 to adjust the pH to 2.0, and the pH was adjusted to 1 at 37 ° C.
It was left for an hour (acid-treated product). After that, the acid-treated product was divided into two parts, and a part thereof was added with 0.04 mg of pepsin (manufactured by Difico, product number 0151-17) and reacted at 37 ° C. for 1 hour (pepsin-treated product). The rest are
After adjusting the pH to 7.0 with NaOH, the mixture was further divided into two, and 1.4 mg of amylase (manufactured by Sigma, product number A-0521) and 1.7 mg of lipase (manufactured by Sigma, product number L-3126) were added per 1 g. In addition, 37
The reaction was carried out at 0 ° C for 1 hour (amylase-treated product, lipase-treated product).

In the same manner as in Test Example 1, the lactic acid bacterium growth promoting effect of 5 samples containing the above-mentioned 4 treated products and the water extract of Example 1 as a control was examined.

The results are shown in Table 3, and the effect of promoting the growth of lactic acid bacteria by the acid-treated product and the digestive enzyme-treated product was exactly the same as that of the control.

[0036]

[Table 3]

From this, it is considered that the lactic acid bacterium growth promoting substance of the present invention reaches the large intestine, which is the habitat of lactic acid bacteria, while maintaining its activity.

Test Example 3 Using 8 healthy adult men and women aged 22 to 33 as test subjects, 0.8 g of the water extract of Example 1 was continuously administered after morning, noon and dinner 14
It was ingested orally for a day. During this period, immediately before the start of intake (day 0), 1 week after the start of intake (7th day), 2 weeks after the start of intake (14th day), and 2 weeks after the end of intake (28th day), the feces of each person Were collected, and the intestinal flora was searched according to the method of Mitsuoka (Michioka Tomohi, "World of Enterobacteriaceae", Sogyosha, Tokyo, p53-65).

FIG. 2 shows the changes in the intestinal flora on average for 8 persons. The beneficial bacteria Bifidobacterium, L
actobacillus increased 1 to 2 weeks after the start of intake, and Clostridium per
fringens, C.I. dificile decreased.

As a result, Bifidob occupies the total number of bacteria
The percentage of acterium is 49.5% (just before the start of intake)
To 62.5% (2 weeks after ingestion).

FIG. 3 shows the changes in the number of Bifidobacterium bacteria immediately before ingestion of the test subjects and two weeks after the ingestion. An increase in the number of bacteria was observed in 7 subjects except 1 subject.

As described above, the lactic acid bacterium growth-promoting substance of the present invention increases bacteria of the genus Bifidobacterium and Lactobacillus, which are useful enteric bacteria,
It was shown to have an intestinal flora improving effect of reducing Clostridium bacterium, which is a harmful bacterium.

Test Example 4 The water extract of Example 1 was subjected to an ultrafiltration membrane having a molecular weight cut off of 10,000 to obtain a low molecular weight fraction of 10,000 or less and 10
The effect of each fraction on the growth of lactic acid bacteria was investigated by dividing it into more than 000 high molecular weight fractions. The test was performed by the method of Test Example 1.

The results are shown in Table 4. The low molecular weight fraction promoted the growth of all lactic acid bacteria, whereas the high molecular weight fraction showed 2
Only the strain showed an effect, the degree of which was very weak.

[0045]

[Table 4]

From this, it is estimated that the lactic acid bacterium growth promoting factor contained in the water extract of Example 1 is a low molecular weight substance having a molecular weight of less than 10,000.

[0047]

The effects of the present invention are as follows. 1) By ingesting the lactic acid bacteria growth-promoting substance of the present invention, bacteria belonging to the genus Bifidobacterium and Lactobacillus can be selectively grown, and intestinal flora in which beneficial bacteria are dominant can be formed. 2) The amount of beneficial bacteria in the intestine can be increased by ingesting about 2.4 g a day as a crude extract. 3) Since this substance has no odor specific to cocoa beans and is odorless, it can be added to various foods. 4) Since it is extracted from the conventionally discarded cacao husk, it can be produced at low cost. 5) Since this substance is stable to acids and digestive enzymes, it reaches the large intestine, which is the habitat of beneficial bacteria, without losing its activity. 6) In the production of fermented milk, the effect of shortening the fermentation period and simplifying the fermentation operation can be obtained by adding the present growth promoting substance.

[Brief description of drawings]

FIG. 1 is a treatment system diagram showing a method for treating cocoa husk water extract with acid and digestive enzymes.

FIG. 2 is a characteristic diagram showing changes in intestinal flora due to ingestion of cacao husk water extract.

FIG. 3 is a characteristic diagram showing changes in the number of Bifidobacterium among 8 test subjects.

─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Masanori Ito 1-2-1-803 Satsukidai, Misato City, Saitama Prefecture (72) Inventor Tatsuya Uewaki 94-1-407 (56) White hoe, Urawa City, Saitama Prefecture (56) References HEI 6-217733 (JP, A) JP-A H6-125771 (JP, A) (58) Fields investigated (Int.Cl. ⁷ , DB name) C12N 1/00-1/38 JSTPlus (JOIS) BIOSIS / WPI (DIALOG)

Claims (3)

(57) [Claims] 1. A lactic acid bacterium growth promoting substance comprising, as an active ingredient, an extraction residue of an organic solvent which is compatible with water of cacao bean and / or cacao husk which is an outer coat of cacao bean. 2. The lactic acid bacterium growth promoting substance according to claim 1, wherein a component having a molecular weight of 10,000 or less in the water extract is used as an active ingredient. 3. The lactic acid bacterium growth promoting substance according to claim 1, wherein the lactic acid bacterium that grows is a bacterium belonging to the genus Bifidobacterium or Lactobacillus.