Microbial culture medium and microbial culture method using acid/activated clay

The effect of the addition of acid clay or activated clay to the culture medium was examined as follows. Using Sammotsuogonto and Daijokito, which are Kampo extracts as the test samples, Staphylococcus aureus IFO 13276 was added to soybean-casein-digest broth media (hereinafter referred to as SCD media) containing acid clay or activated clay under the conditions listed in Table 1, thereby investigating the growth of the target bacterium in the culture media having different compositions, in accordance with the “Medium Performance Test and Growth Inhibitor Verification Test” under the microbial limit test method described in Japanese Pharmacopoeia. After the proliferating cultivation, the culture was spread and cultured on a Vogel-Johnson agar, and investigated for the qualitative response of the Staphylococcus aureus.

Conventional Example 1

As a conventional example, a culture test was conducted in the same manner as in Examples, except that a medium containing activated carbon was used.

Comparative Examples 1-1 to 1-3

As comparative examples, culture tests were conducted using media containing the test sample and/or the target bacterium without activated carbon and activated clay.

Comparative Examples 1-4 and 1-5

As comparative examples, culture tests were conducted in the same manner as in Examples, except that media containing SEPABEADS SP-700(trademark: manufactured by Mitsubishi Chemical Corporation), which is a typical ion adsorbent, were used.

Cultures showed colony formation and qualitative response were rated as ◯, colony formation only or weak qualitative response as Δ, and no colony formation as x. The results are listed in Table 1.

The results listed in Table 1 indicate that the target bacterium did not grow in the ordinary media containing a Kampo extract as the test sample. On the other hand, in the examples using activated clay-containing media, the target bacterium grew as in the case of the conventional examples using activated carbon (powder). In the examples using acid clay-containing media, the target bacterium grew by the Kampo extracts. Accordingly, it was confirmed that the addition of acid clay or activated clay allows adequate growth of the target bacterium in the antibacterial Kampo extracts. In some examples, the granular activated carbon and ion adsorbent were not effective against the Kampo extracts.

The effect of activated clay-containing culture media with different test samples was examined as follows. Using Gorinsan and Ryutanshakanto, which are Kampo extracts, as the test samples, Staphylococcus aureus IFO 13276 was added to culture media (basal medium: SCD medium) containing activated clay in amounts listed in Table 2 below. The test and evaluation were carried out in the same manner as in Example 1.

Cultures showed colony formation and qualitative response were rated as ◯, colony formation only or weak qualitative response as Δ, and no colony formation as x. The results are listed in Table 2.

The results listed in Table 2 indicate that the addition of activated clay to the culture medium repressed the antibacterial activity of Gorinsan and Ryutanshakanto to allow the growth of the target bacterium.

Conventional Example 3, Example 3, and Comparative Example 3

The effect of culture media containing activated clay and activated carbon was examined as follows. Using Junchoto, Gorinsan, Ryutanshakanto, Tsudosan, and Daijokito, which are Kampo extracts, as the test samples, Staphylococcus aureus IFO 13276 was added to culture media (basal medium: SCD medium) containing activated clay and activated carbon in amounts listed in Table 3 below. The test and evaluation were carried out in the same manner as in Example 1.

Cultures showed colony formation and qualitative response were rated as ◯, colony formation only or weak qualitative response as Δ, and no colony formation as x. The results are listed in Table 3.

The results listed in Table 3 indicate that the target bacterium more markedly grew in the examples using culture media containing activated clay and activated carbon, in comparison with the case using activated carbon alone. It was thus confirmed that the combination of activated clay and activated carbon more effectively represses the antibacterial activity of the test samples.

Using Keishibukuryogan, Yokukansan, and Daikenchuto as the test samples, Staphylococcus aureus IFO 13276 was added to culture media (basal medium: SCD medium) containing activated clay and activated carbon in amounts listed in Table 4 below. The test and evaluation were carried out in the same manner as in Example 1.

Cultures showed colony formation and qualitative response were rated as ◯, colony formation only or weak qualitative response as Δ, and no colony formation as x. The results are listed in Table 4.

The results listed in Table 4 indicate that the qualitative response owing to the favorable growth of the target bacterium was constantly observed in the examples using the culture media containing activated clay and activated carbon, but stable qualitative response was not observed in the cases using activated carbon alone or no additive. It was thus confirmed that the combination of activated clay and activated carbon more effectively represses the antibacterial activity of the test samples, thereby providing stable test results.

Using Keishibukuryogan, Yokukansan, and Daikenchuto as the test samples, Staphylococcus aureus IFO 13276 was added to culture media (basal medium: SCD medium) containing activated clay and activated carbon in amounts listed in Table 5 below. The test and evaluation were carried out in accordance with United States Pharmacopeia 30, <62> MICROBIOLOGICAL EXAMINATION OF NONSTERILE PRODUCTS: TESTS FOR SPECIFIED MICROORGANISMS.

Cultures showed colony formation and qualitative response were rated as ◯, colony formation only or weak qualitative response as Δ, and no colony formation as x. The results are listed in Table 5.

The results listed in Table 5 indicate that the qualitative response owing to the favorable growth of the target bacterium was constantly observed in the examples using the culture media containing activated clay and activated carbon, but stable qualitative response was not observed in the cases using activated carbon alone or no additive. It was thus confirmed that the combination of activated clay and activated carbon more effectively represses the antibacterial activity of the test samples, thereby providing stable test results.

Comparative Example 6, Example 6 and Conventional Example 6

The influence of the growth of bacteria in the culture media each containing 5% by weight of activated carbon (powder) alone or 5% by weight of activated clay alone was examined as follows. According to the “Medium Performance Test and Growth Inhibitor Verification Test” under the microbial limit test method described in Japanese Pharmacopoeia, the growth of target bacteria in SCD media alone and SCD media containing 5% by weight of activated clay or activated carbon was investigated. The bacterial species tested were Staphylococcus aureus IFO 13276 and Pseudomonas aeruginosa IFO 13275. After the proliferating cultivation, the cultures of the Staphylococcus aureus were spread and cultured on Vogel-Johnson agar and the cultures of the Pseudomonas aeruginosa were spread and cultured on NAC agar media, respectively, and were investigated for the qualitative response of Staphylococcus aureus or Pseudomonas aeruginosa.

Cultures showed colony formation and qualitative response were rated as ◯, colony formation only or weak qualitative response as Δ, and no colony formation as x. Ten samples were tested in each of the examples, comparative examples, and conventional examples. The results are listed in Table 6 below.

The results listed in Table 6 indicate that the colony formation of Staphylococcus aureus was weak on some media containing 5% by weight of activated carbon. This is likely due to that excessive portions of activated carbon adsorb the components of the media to adversely affect the microbial growth. On the other hand, the addition of activated clay gave no adverse effect, even when the loading was increased to 10% by weight.

The influences of activated carbon and activated clay on culture media were closely examined as follows. According to the microbial limit test method described in Japanese Pharmacopoeia, the bacterial concentrations in SCD media alone and SCD media containing activated clay and/or activated carbon in amounts listed in Table 7 below were measured after shaking culture for 24 hours. As the target bacteria, Staphylococcus aureus IFO 13276 and Pseudomonas aeruginosa IFO 13275 were used. The initial number of the bacteria was about 100 cfu. The results are listed in Table 7, wherein “%” refers to “% by weight”.

The results listed in Table 7 indicate that the proliferation potencies of Staphylococcus aureus and Pseudomonas aeruginosa in the media containing 3% by weight of activated carbon and 5% by weight of activated clay were about the same as those in the media with no additive. On the other hand, the proliferation potency of Staphylococcus aureus was particularly low in the media containing activated carbon alone, indicating that the addition of activated clay suppresses the deterioration of the proliferation potency caused by the addition of activated carbon.

Using Keishibukuryogan as the test sample, under the assumption of microbial limit test described in United States Pharmacopeia, formation of typical colonies in test sample solutions was examined in accordance with United States Pharmacopeia 28, <61> MICROBIAL LIMIT TESTS. The test sample solutions were a SCD medium containing 3% by weight of activated carbon and 5% by weight of activated clay (ISCD medium), a SCD medium with no additive, and a SCD medium containing 0.5% by weight of lecithin and 4.0% by weight of polysorbate 20 (LPSCD medium), the sample concentration during cultivation was 0.01 g/mL, and the target bacterium was Staphylococcus aureus ATCC 6538.

Cultures showed colony formation and qualitative response were rated as ◯, colony formation only or weak qualitative response as Δ, and no colony formation as x. The results are listed in Table 8 below.

The results listed in Table 8 indicate that no typical colony was formed in the culture using the LPSCD medium, which is an inactivated culture medium included in United States Pharmacopeia, as the test sample solution, suggesting that the influence of antibacterial substances was not repressed. On the other hand, typical colonies were formed in the culture using the ISCD medium containing activated clay and activated carbon, suggesting that the influence of antibacterial substances was repressed.

Using Keishibukuryogan as the test sample, Salmonella enterica spp. enterica serotype abony ACM 5080 was added to the media (basal medium: SCD medium) containing activated clay and activated carbon in amounts listed in Table 9 below. The test and evaluation were carried out in accordance with United States Pharmacopeia 30, <62> MICROBIOLOGICAL EXAMINATION OF NONSTERILE PRODUCTS: TESTS FOR SPECIFIED MICROORGANISMS.

Cultures showed colony formation and qualitative response were rated as ◯, colony formation only or weak qualitative response as Δ, and no colony formation as x. The results are listed in Table 9.

The results listed in Table 9 indicate that the target bacterium grew in the culture media of examples containing activated clay and activated carbon, but they did not grow in the media containing activated carbon alone. These facts suggest that the combination of activated clay and activated carbon more effectively represses the antibacterial activity of the test sample.

The growth of a target bacterium in media containing a food as the test sample was examined as follows, wherein the test sample was Japanese horseradish or tea leaves. 10 g, 1 g, and 0.1 g of a Japanese horseradish paste or tea leaves, (which had been subjected to autoclave sterilization to prevent contamination), were added to a peptone buffer respectively thereby making 100 mL of 10-fold, 100-fold, and 1000-fold diluted test sample solutions. 10 mL of the respective test sample solutions was individually added to 90 mL portions of SCD media, SCD media containing lecithin and polysorbate (SCDLP medium), and SCD media containing 3% by weight of activated carbon and 5% by weight of activated clay (ISCD medium), and the growth of the test bacterium was investigated in accordance with the “Medium Performance Test and Growth Inhibitor Verification Test” under the microbial limit test method described in Japanese Pharmacopoeia. The bacterium tested was Staphylococcus aureus IFO 13276, and the test and evaluation were carried out in the same manner as in Example 1.

Cultures showed colony formation and qualitative response were rated as ◯, colony formation only or weak qualitative response as Δ, and no colony formation as x. The results are listed in Table 10 below.

The results listed in Table 10 indicate that the target bacterium formed no colony in the SCD and SCDLP media containing the 10-fold diluted solutions of Japanese horseradish and tea leaves with high concentration, suggesting that the influence of antibacterial substances was not repressed. On the other hand, colonies were formed in all the mixtures of the diluted solutions and the ISCD media containing activated clay and activated carbon, suggesting that the influence of antibacterial substances was repressed. These facts indicate that the culture medium of the present invention adsorbs and reduces antibacterial substances contained in Japanese horseradish and tea leaves as well as those contained in Kampo extracts, thereby repressing their antibacterial activity. Accordingly, the culture medium is applicable to the improvement of food microbial test.

The growth of a target bacterium in media containing a dry spice as the test sample was examined as follows. 10 g, 5 g, and 0.5 g of turmeric powder, pepper powder, and dry basil leaves were made into 100 mL solutions, from which 10-fold, 20-fold, and 200-fold diluted solutions were prepared. 10 mL of the respective test sample solutions were individually added to 90 mL portions of SCD media containing 7.5% of salt, which are listed in the microbial limit test method for crude drugs described in Japanese Pharmacopoeia, SCD media containing 7.5% of salt, 3% by weight of activated carbon, and 5% by weight of activated clay (SCDAA media containing 7.5% of salt), and SCDB media containing 7.5% of salt and 5% by weight of activated clay (SCDA media containing 7.5% of salt). The growth of the test bacterium was investigated in accordance with the “Medium Performance Test and Growth Inhibitor Verification Test” under the microbial limit test method described in Japanese Pharmacopoeia. The bacterium tested was Staphylococcus aureus IFO 13276, and the test and evaluation were carried out in the same manner as in Example 1.

Cultures showed colony formation and qualitative response were rated as ◯, colony formation only or weak qualitative response as Δ, and no colony formation as x. The results are listed in Table 11 below.

The results listed in Table 11 indicate that the target bacterium formed no colony in the SCD media containing 7.5% of salt and the 10-fold or 20-fold diluted solution of turmeric powder, pepper powder, or dry basil leaves, suggesting that the influence of antibacterial substances was not repressed. On the other hand, colonies were formed in all the mixtures of the dry basil leaves diluted solutions in the SODA media containing 7.5% of salt and activated clay and all the mixtures in the SCDAA media containing 7.5% of salt, activated clay, and activated carbon, suggesting that the influence of antibacterial substances was repressed. These facts indicate that the culture medium of the present invention adsorbs and reduces antibacterial substances contained in a dry spice as well as those contained in Kampo extracts, thereby inhibiting their antibacterial activity. Accordingly, the culture medium is applicable to the improvement of food microbial test.

The growth of target bacteria in media containing an antibiotic as the test sample was examined as follows. Chloramphenicol was added to SCD media which are described in the microbial limit test method in Japanese Pharmacopoeia, SCD media containing 3% by weight of activated carbon and 5% by weight of activated clay (ISCD media), lactose broth media (hereinafter referred to as LB media), and LB media containing 3% by weight of activated carbon and 5% by weight of activated clay (ILB media) at concentrations of 250 mg/L, 50 mg/L, 5 mg/L, 0.5 mg/L, and 0.05 mg/L respectively. The growth of the test bacteria was investigated in accordance with the “Medium Performance Test and Growth Inhibitor Verification Test” under the microbial limit test method described in Japanese Pharmacopoeia. The bacteria tested were Staphylococcus aureus IFO 13276 for the SCD and ISCD media, and Escherichia coli IFO 3972 for the LB and ILB media. After the proliferating cultivation, the cultures of the Staphylococcus aureus and Escherichia coli were spread and cultured on Vogel-Johnson agar media ( Staphylococcus aureus ) and MacConkey agar media ( Escherichia coli ), respectively, and investigated for the qualitative response.

Cultures showed colony formation and qualitative response were rated as ◯, colony formation only or weak qualitative response as Δ, and no colony formation as x. The results are listed in Table 12 below, wherein “%” refers to “% by weight”.

The results listed in Table 12 indicate that Staphylococcus aureus and Escherichia coli formed colonies and showed qualitative responses after cultivation in the ISCD and ILB media containing antibiotics at concentrations 100 times higher than ordinary SCD and LB media, suggesting that the influence of the antibiotics was repressed. These facts indicate that the culture medium of the present invention represses the antibacterial activity of samples containing antibiotics as well as that of Kampo extracts. Accordingly, the culture medium is applicable to the improvement of microbial test on general drugs.

The microbial growth in media containing activated clay and activated carbon at a ratio of 1% or 2% of activated carbon with reference to 5% of activated clay was examined as follows. Using Junchoto, Gorinsan, Ryutanshakanto, Tsudosan, and Daijokito, which are Kampo extracts, as the test samples, the test and evaluation were carried out in the same manner as in Example 3.

Cultures showed colony formation and qualitative response were rated as ◯, colony formation only or weak qualitative response as Δ, and no colony formation as x. The results are listed in Table 13 below.

The results listed in Table 13 indicate that the target bacterium sufficiently grew in the media containing 5% by weight of activated clay and activated carbon even at a content of 1% by weight, suggesting that the antibacterial activity of the test samples was effectively repressed.