JP2010195711A - Biofilm formation promoter - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a biofilm formation promoter which has action of promoting biofilm formation by lactic acid bacteria and can be used as a pharmaceutical drug, food, a food additive, an industrial additive, etc. <P>SOLUTION: The biofilm formation promoter of at least one lactic acid bacteria chosen from lactic acid bacillus and lactic acid cocci of the genera Leuconostoc and Pediococcus contains fructose. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a biofilm formation promoter for lactic acid bacteria.

A biofilm is a community of microorganisms formed by microorganisms such as bacteria and molds adhering to a solid or liquid surface together with secretions. Microorganisms often form biofilms when they inhabit the environment or in the human body, and when microbes form biofilms, they are in a floating state against stress such as cleaning removal, drugs, and heat. It is known that it will be stronger.
For such microbial biofilms, diseases are caused by the formation of human tissues such as skin, and biofilms formed on fresh food products such as vegetables, raw materials for processed foods and cooking utensils are rotted, deteriorated and food poisoning It is known to cause

On the other hand, microbial biofilms have a beneficial effect on the medical scene and industry, and are used industrially. For example, water purification by membrane biofilm reactor, purification of petroleum-contaminated marine environment by extracellular polysaccharide, efficiency of oil resolution by microorganisms using quorum sensing (information transmission between microorganism cells), bioremediation by biofilm on plant surface As an example, promotion of substance production by fermentation bacterial biofilm formation on a solid surface can be mentioned.
Biofilms are also applied to environmentally low-load products such as biofilms (biojelly) as environmentally-friendly ship bottom paints; hydroponics of organic fertilizers using root biofilms; Examples thereof include highly functional biopolymers such as water-absorbing water-retaining biopolymers and oil-water separating biopolymers.

Examples of a method for promoting the formation of the microbial biofilm include, for example, a method for promoting biofilm formation of Pseudomonas bacteria using a natural material such as wood chips as an attachment carrier (Patent Document 1), and a marine by using a new butenolide. A bacterial biofilm formation promoting method (Patent Document 2) is known.

  By the way, lactic acid bacteria have been used for producing foods such as fermented milk, lactic acid bacteria beverages, and dairy products such as fermented butter for a long time, but lactic acid bacteria themselves have various pharmacological effects such as intestinal regulation effects, or Because it secretes substances having antibacterial effects such as bacteriocin, it is used as a material for health foods and pharmaceuticals. These lactic acid bacteria are known to act more effectively when attached to some kind of carrier. For example, improvement of the intestinal environment by adhesion of useful bacteria in the digestive tract to host cells (Non-patent Document 1), lactic acid bacteria Application to medical treatment such as prevention of bacterial infection by adhesion to the urogenital tract and intestinal tract (Non-patent Documents 2 and 3) has been reported, and application to lactic acid fermentation reactors as an effective industrial use (Non-patent Document 4) ) Has also been reported.

In the biofilm of lactic acid bacteria, Streptococcus mutans is produced by supplying sucrose as a single carbon source for Streptococcus, which is a carious bacterium, so that EPS (extracellular polysaccharide), lipid, Forming many biofilms containing a lot of nucleic acids (Non-patent Document 5), in the presence of an excess of carbon sources such as sucrose, glucose, fructose, etc. under low nitrogen conditions , the biofilm in Streptococcus gordonii There are reports that the number of bacteria in the film increases (Non-patent Document 6), and that in oral plaque, the number of Streptococcus spp. In the plaque increases when both sucrose or glucose and fructose are added. (Non-Patent Documents 7 and 8).
In addition, in Enterococcus faecalis , an enterococci , it has been reported that when glucose, maltose, mannose and fructose are used as a carbon source, the amount of biofilm formation is larger than sucrose, lactose and trehalose ( Non-patent document 9).
On the other hand, in lactobacilli, the addition of sucrose is known to increase the number of bacteria of the genus Lactobasillus in the oral plaque (Non-patent Documents 7 and 8) and is related to the amount of biofilm formation. There are no reports.

  Fructose is used as a microbial culture medium additive and food additive, and also promotes the growth of microorganisms during liquid culture even in lactic acid bacteria (Patent Documents 3 to 5). Both fructose and glucose are added. In some cases, it has been reported that the number of Lactobacillus species in the oral plaque increases (Non-patent Documents 7 and 8).

  However, it has not been known so far whether fructose affects the formation of lactobacilli and biofilms of lactic acid cocci other than caries and enterococci.

JP 2005-313159 A JP-A-6-135954 JP 2004-215561 A Japanese Patent Laid-Open No. 61-231966 JP 2007-31345 A

FEMS Immunology and Medical Microbioloby 26 pp137-142 (1999) Infection Disease in Obstetrics and Gynecolology volume 2008 Article ID 549640 (2008) In Proceedings of Lactic '94 .pp201-212 (1994) Applied and Enbironmental Microbiology 63 (7) pp2533-2542 (1997) Applied and Enviornmental Microbiology 72 (10) 6734-6742 (2006) Infection and Immunity 71 (8) 4759-4766 (2003) Caries Reaserch 41 (5) 406-412 (2007) Caries Reasearch 40 (6) 546-549 (2006) BMC Microbiology 6 (60) 1-8 (2006)

  The present invention relates to providing a biofilm formation accelerator that has an action of promoting the formation of lactic acid bacteria biofilm and can be used as a pharmaceutical, food, food additive, industrial additive, and the like.

  When the present inventors examined the biofilm formation of lactic acid bacteria, they found that fructose significantly promotes the biofilm formation of specific lactic acid bacteria.

  That is, the present invention relates to a biofilm formation promoter of one or more lactic acid bacteria selected from lactobacilli and lactic acid bacteria other than enterococci and fructose.

  The biofilm formation promoter for lactic acid bacteria of the present invention has the effect of significantly promoting the biofilm formation of lactobacilli other than lactobacilli or caries and enterococci. Therefore, according to the present invention, improvement of the intestinal environment, adjustment of the intestine, prevention of bacterial infection of the urogenital tract, intestinal tract, etc., pharmaceuticals, quasi drugs, various foods and drinks, food additives for promoting lactic acid fermentation Industrial additives and pet food can be provided.

  Fructose may be used as it is produced by general chemical synthesis, produced by fermentation, isolated or purified from natural products, or natural products containing these. For example, the thing derived from vegetables, such as a tomato, a carrot, an onion, can also be used. Fructose may be used alone or in combination with at least one other saccharide.

Here, in the present invention, the promotion of biofilm formation means promoting the formation of a community (biofilm) formed by lactic acid bacteria together with secretions and deposits. Biofilm formation is a process in which bacteria attached to the surface of the carrier in the early stages of formation gradually increase the number of inhabitants while repeating colonization and detachment, and co-communicate with secretions and deposits. The film formation promoting action is different from the microorganism growth promoting action.
The lactic acid bacteria involved in the lactic acid bacteria of biofilm formation promoting present invention, dental caries bacteria and lactic streptococci other than Enterococci, is a Lactobacillus, Lactobacillus, Leuconostoc (Leuconostoc) genus Lactococcus, Pediococcus (Pediococcus The genus Lactococcus and Lactococcus are preferred. The lactobacilli are preferably Lactobacilus genus lactobacilli and Bifidobacterium genus lactobacilli.

Specific examples of Lactobacillus genus Lactobacillus include Lactobacillus plantarum , Lactobacilus gasseri , Lactobacillus brevis , Lactobacillus fructivorans, Lactobacillus fructivorans, and Lactobacillus fructivorans. (Lactobacilus acetotolerans), Lactobacillus acidophilus (Lactobacilus acidophilus), Lactobacillus Buchineri (Lactobacillus buchneri), Lactobacillus del Burekki subsp del Burekki (Lactobacilus delbrueckii subsp. delbrueckii), Lactobacillus del Burekki subsp. bulgaricus (Lactobacilus delbrueckii subsp. bulgaricus), Lactobacillus del Burekki subsp. lactis (Lactobacilus delbrueckii subsp. lactis), Kutobachirusu fermentum (Lactobacilus fermentum), Lactobacillus Furukuchiboransu (Lactobacillus fructivorans), Lactobacillus Homohiochi (Lactobacillus homohiochii), Lactobacillus kefir (Lactobacillus kefir), Lactobacillus Rindoneri (Lactobacillus rindneri), Lactobacillus Parabuchineri (Lactobacillus parabuchneri), Lactobacillus paracasei subsp. paracasei (Lactobacillus paracasei subsp. paracasei), Lactobacillus paracasei subsp tolerance (Lactobacillus paracasei subsp. tolerans), Lactobacillus Parakefia (Lactobacillus parakefir), Lactobacillus pentosus (Lactobacillus pentosus), Lactobacillus reuteri (Lactobacillus reuteri), Lactobacillus salmon ( Lactobacillus sake ), Lactobacillus salvary Usu subsp Sarishiniusu (Lactobacillus salivarius subsp. Salicinius), Lactobacillus Sarubariusu subsp Sarubariusu (Lactobacillus salivarius subsp. Salivarius), Lactobacillus Vaginarisu (Lactobacilus vaginalis), Lactobacillus rhamnosus (Lactobacilus rhamnosus), Lactobacillus casei (Lactobacilus casei), Lactobacillus Ajirisu (Lactobacilus agilis), Lactobacillus alimentarius (Lactobacilus alimentarius), Lactobacillus Amirofirasu (Lactobacilus amylophilus), Lactobacillus amylovorus (Lactobacilus amylovorus), Lactobacillus animalis (Lactobacilus animalis), Lactobacillus Abiariusu subsp Arafinosasu (Lactobacilus aviarius subsp. araffinosus ), Lactobacillus avirarius subs Lactobacilus aviarius subsp. Aviarius , Lactobacillus bifermentans , Lactobacillus catenaformis , Lactobacillus collinoides ( Lactobacillus collinoides ), Lactobacillus subinos Lactobacilus coryniformis subsp. coryniformis), Lactobacillus Korini Four miss subsp Torukuensu (Lactobacilus coryniformis subsp. torquens), Lactobacillus crispatus (Lactobacillus crispatus), Lactobacillus Kabatasu (Lactobacillus curvatus), Lactobacillus Fashiminisu (Lactobacilus farciminis), Lactobacillus Furukutosasu (Lactobacillus fructosus), Lactobacillus Garinaramu (Lactobacillus galinarum), lacto Chirusu graminis (Lactobacillus graminis), Lactobacillus Hamusuteri (Lactobacillus hamsteri), Lactobacillus helveticus (Lactobacillus helveticus), Lactobacillus Hirugadi (Lactobacillus hilgardii), Lactobacillus Intel stay Naris (Lactobacillus intestinalis), Lactobacillus Jensenii (Lactobacillus jensenii), lacto Bacillus johnsonii (Lactobacillus johnsonii), Lactobacillus Kefi Rano tumefaciens (Lactobacillus kefiranofaciens), Lactobacillus Kefiagunamu (Lactobacillus kefirgranum), Lactobacillus Marais fermentans (Lactobacillus malefermentans), Lactobacillus Mali (Lactobacillus mali), Lactobacillus Marutaromikasu (Lactobacillus maltaromicus), Lactobacillus murinus (Lactobacillus murinus), Rakutobachi Scan Oris (Lactobacillus oris), Lactobacillus Pontis (Lactobacillus pontis), Lactobacillus Ruminisu (Lactobacillus ruminis), Lactobacillus San Francisco (Lactobacilus sunfrancisco), Lactobacillus Shapeae (Lactobacilus sharpeae), Lactobacillus Suebikasu (Lactobacilus suebicus), Lactobacillus Bitsurinasu ( Lactobacilus vitulinus ) and the like.

Specific examples of Bifidobacterium genus Lactobacillus include Bifidobacterium bifidum , Bifidobacterium longum , Bifidobacterium breve , Bifidobacterium um Anguatamu (Bifidobacterium angulatum), Bifidobacterium animalis (Bifidobacterium animalis), Bifidobacterium asteroides (Bifidobacterium asteroides), Bifidobacterium Boumu (Bifidobacterium boum), Bifidobacterium Katenaratamu (Bifidobacterium catenulatum), Bifidobakuteri Umm Koerinamu (Bifidobacterium choerinum), Bifidobacterium Corynebacterium form (Bifidobacterium coryneforme), Bifidobacterium Kunikari (Bifidobacterium cuniculi), Bifidobaku Potassium Dentiumu (Bifidobacterium dentium), Bifidobacterium Garikamu (Bifidobacterium gallicum), Bifidobacterium Garinaramu (Bifidobacterium gallinarum), Bifidobacterium Indiana cam (Bifidobacterium indicum), Bifidobacterium magnum (Bifidobacterium magnum), Bifido bacterium Merishikamu (Bifidobacterium merycicum), Bifidobacterium minimum (Bifidobacterium minimum), Bifidobacterium shoe de Catena La Tam (Bifidobacterium pseudocatenulatum), Bifidobacterium shoe Delon gum (Bifidobacterium pseudolongum), Bifidobacterium Shikuraerofi Rum ( Bifidobacterium psychraerophilum ), Bifidobacterium prolum ( Bifidobacterium pullorum ), Bifidobacterium ruminantium ( Bifidobacterium rumin antium ), Bifidobacterium saeculare , Bifidobacterium scardovii , Bifidobacterium simiae , Bifidobacterium subtile , Bifidobacterium subma ram (Bifidobacterium thermacidophilum), Bifidobacterium thermophilum (Bifidobacterium thermophilum), Bifidobacterium Urinarisu (Bifidobacterium urinalis) Bifidobacterium (Bifidobacterium), etc., and the like.

In addition, preferable specific examples of the above leuconostock lactic acid cocci include Leuconostoc mesenteroides , Leuconostoc citreum , Leuconostoc lactis , Leuconostoc lactis and Leuconostoc lactis. Tinamu (Leuconostoc argentinum), Leuconostoc Kanosamu (Leuconostoc carnosum), Leuconostoc file Lux (Leuconostoc fallax), Leuconostoc Geridamu (Leuconostoc gelidum), and the like Leuconostoc shoe domain initiative Roy Death (Leuconostoc psudomesenteriodes), etc. .

Preferred examples of the above-mentioned genus Lactococci of Pediococcus include Pediococcus pentosaceus , Pediococcus acidilactici , Pediococcus damnosus , Pediococcus dediocuscus ( Pediococcus dextrinicus ), Pediococcus inopinatus , Pediococcus parvulus , Pediococcus urinaeequi and the like.

Specific examples of suitable Lactococcus lactic acid cocci include Lactococcus lactis .

  Among these lactic acid bacteria, lactobacilli, leucostock lactic acid cocci, and pediococcus lactic acid cocci are preferable, lactobacilli lactic acid bacillus and pediococcus pentosaceus are more preferable, lactobacilli lactic acid bacillus are more preferable, and biofilm formation In terms of promotion, Lactobacillus plantarum and Lactobacillus gasseri are particularly preferable.

  The fructose of this invention has the effect | action which accelerates | stimulates significantly the biofilm formation of Lactobacillus, Leuconostoc genus Lactococcus, and Pediococcus genus Lactococcus as shown in Example 1 and 2 mentioned later. Therefore, the fructose of the present invention can be used as a biofilm formation accelerator for the lactic acid bacteria, and can be used for producing the biofilm formation accelerator.

By using the biofilm formation promoter of lactic acid bacteria of the present invention, it is possible to improve the intestinal environment, prevent intestinal regulation, infection of bacteria in the genitourinary tract, intestinal tract and the like, and promote lactic acid fermentation.
Therefore, the biofilm formation promoter of the lactic acid bacterium of the present invention is a pharmaceutical, quasi-drug for the purpose of improving the intestinal environment, preventing the infection of bacteria in the intestine, urogenital tract, intestinal tract, etc., and promoting lactic acid fermentation, It can be used as various foods, food additives, industrial additives and pet food.
The biofilm formation promoter for lactic acid bacteria of the present invention is, for example, administered to humans or animals, and sprayed, sprayed, applied, or ingested in various environments where biofilm formation is desired, such as vegetables, intestinal walls, and fermenters. It can be used in the way.

When the biofilm formation promoter of the present invention is used as a pharmaceutical, it can be administered in any dosage form. Examples of the dosage form include oral, enteral, transmucosal, and injection, and oral administration is preferred.
Examples of the dosage form of the preparation for oral administration include tablets, coated tablets, capsules, granules, powders, powders, sustained-release preparations, suspensions, emulsions, oral liquids, dragees, pills, fine granules Agents, syrups, elixirs and the like. Examples of parenteral administration include intravenous injection, intramuscular injection, inhalation, infusion solution, suppository, inhalant, spray, aerosol, coating agent, transdermal absorption agent, eye drop, and nasal drop.

  In the above preparation, a pharmaceutically acceptable carrier can be added to fructose. Such carriers include, for example, excipients, binders, disintegrants, lubricants, diluents, osmotic pressure regulators, fluidity promoters, absorption aids, pH adjusters, emulsifiers, preservatives, stabilization. Agent, antioxidant, colorant, UV absorber, wetting agent, thickener, brightener, activity enhancer, anti-inflammatory agent, bactericidal agent, flavoring agent, flavoring agent, extender, surfactant, dispersant, Buffering agents, preservatives, fixing agents, fragrances, coating agents, propellants and the like can be mentioned.

  Moreover, a nitrogen compound, an amino acid, a vitamin, etc., various medicinal ingredients, etc. can be further mix | blended with the said formulation.

  Examples of nitrogen compounds include yeast extract; meat extract; natural nitrogen sources such as peptone and soyton; inorganic nitrogen compounds such as ammonium salts such as ammonium chloride, ammonium sulfate and ammonium phosphate.

  Examples of amino acids include arginine, alanine, aspartic acid, cysteine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, asparagine and the like.

  Examples of vitamins include biotin, folic acid, pyridoxal, riboflavin, thiamine, vitamin B12, deoxyriboside, oleic acid and the like.

  The content of fructose in the above preparation is not particularly limited, and it may be a normal dose and may be an amount at which fructose can exert an effect at the action point. In this case, it is about 0.01 to 100% by mass, preferably 0.03 to 100% by mass of the total mass of the preparation.

  When the biofilm formation promoter of the present invention is used as a pharmaceutical, the dosage may vary according to the patient's condition, weight, sex, age or other factors, but the daily dose per adult for oral administration The dosage is usually preferably 0.5 to 5 g as fructose. Moreover, although the said formulation can be administered according to arbitrary administration schedules, it is preferable to administer once to several times a day.

  When using the biofilm formation promoter of lactic acid bacteria of the present invention as various foods and drinks, in addition to general foods and drinks, it is necessary to improve the intestinal environment, prevent intestinal infection, prevent infection of bacteria in the genitourinary tract, etc. Accordingly, it can be applied to functional foods and drinks such as beauty foods and drinks, sick foods and drinks, nutritional functional foods and drinks for specific health, and the like.

  The form of the food or drink may be solid, semi-solid or liquid. Examples of food and drink include confectionery such as breads, noodles, cookies, jelly products, dairy products, frozen foods, instant foods, processed starch products, processed meat products, other processed foods, beverages such as coffee drinks, soups , Seasonings, dietary supplements, and the like, and their ingredients. Further, like the above-mentioned oral administration preparation, it may be in tablet form, pill form, capsule form, liquid form, syrup form, powder form, granule form and the like.

  In addition to fructose, other forms of food and drink, such as foods and drinks, solvents, softeners, oils, emulsifiers, preservatives, fragrances, stabilizers, colorants, ultraviolet absorbers, antioxidants, humectants , Thickeners, sticking agents, dispersants, wetting agents, and nitrogen compounds, amino acids, vitamins, and the like similar to those described above may be mixed and prepared as appropriate.

  In addition, the content of fructose in the food and drink is not particularly limited, and it may be a normal intake amount that is sufficient for fructose to exert its effect at the action point. In this form, it is about 0.01 to 50% by mass, preferably 0.03 to 50% by mass. Moreover, in solid food forms, such as a tablet and processed food, it is about 0.01-70 mass%, and 0.03-70 mass% is preferable.

  When the biofilm formation promoter of lactic acid bacteria of the present invention is used as an industrial additive, the concept is to promote lactic acid fermentation, etc., and powders, tablets, sprays, sprays, oils, which are indicated as necessary , Emulsions, wettable powders, coating agents, aerosols.

  The above industrial additives include, in addition to fructose, other industrial additives, solvents, oils, emulsifiers, preservatives, fragrances, stabilizers, colorants, UV absorbers, antioxidants, thickeners, fixing An agent, a dispersant, a wetting agent, an antibacterial agent, an antifoaming agent, and the like can be blended and prepared as appropriate.

  Moreover, content of fructose in an industrial additive is not specifically limited, Although it changes with the usage forms, it is 0.01-100 mass% normally, and 0.03-100 mass% is preferable.

Example 1 Lactobacillus plantarum promotes biofilm formation Sugars fructose (Wako Pure Chemical Industries), sucrose (Wako Pure Chemical Industries), galactose (Wako Pure Chemical Industries), maltose (Wako Pure Chemical Industries), and glucose (Wako Pure Chemical Industries) were used as controls.

2. Test strain Lactobacillus plantarum (Lactobacillus plantarum) JCM1149 (Japan Collection of Microorganisms), experiments were carried out using Lactobacillus plantarum (Lactobacillus plantarum) M606 Environmental isolates). Lactobacillus plantarum M606 is a strain isolated from onion by an enrichment culture method using MRS medium (OXOID), and a strain having similar properties can be easily separated from onion.

3. Experimental method 10 ml MRS liquid test tube medium (peptone (OXOID) 10.0 g, Lab-lemco Powder (OXOID) 8.0 g, Yeast extract (OXOID) 4.0 g, glucose 20.0 g, Tween80 1 ml, K ₂ HPO ₄ 2.0 g, CH ₃ COONa · 3H ₂ O 5.0 g, Citric Acid Triammonium 2.0 g, MgSO ₄ · 7H ₂ O 0.2 g, MnSO ₄ · 4H ₂ O 0.05 g (54 g / l) pH 6.2) The solution was diluted 10-fold after 24 hours at a temperature of 24 ° C. and used as a test bacterial solution.

1) Preparation of biofilm and evaluation of formation promotion effect were performed as follows.
Using a 24-well microplate (flat bottom) made of PVC (polyvinyl alcohol chloride), in a container with one cover glass (for microscope observation) per well, 0.11 M After dispensing a liquid medium (1000 μl / well) to which saccharides (glucose, fructose, galactose, maltose, sucrose) have been added, add the test bacteria solution (100 μl / well), and cover the container with a lid at 35 ° C. Cultivation was performed for ˜48 hours.
After removing the culture solution, the attached cells and secretions and deposits are made into biofilm, 0.1% crystal violet aqueous solution (Crystal Violet (Kanto Chemical)) is added (1000μL / well), and the biofilm is stained. did.
After removing the crystal violet aqueous solution and washing twice with tap water (1000 μl / well), the biofilm adhered and remaining on the glass was quantified.
The biofilm was quantified by measuring the absorbance (595 nm) of the extract obtained by extracting crystal violet from the stained biofilm using 99.5% ethanol (1000 μL / well).

2) On the other hand, the amount of cells in the biofilm was measured as follows.
The biofilm after the culture was broken by pipetting, and the bacteria in the biofilm were suspended in the culture solution, and the absorbance (600 nm) was measured.
3) The biofilm formation amount was calculated as the biofilm formation rate as follows.
Biofilm formation rate = absorbance measurement value of biofilm dyed extract (measurement wavelength: 595 nm) / absorbance measurement value of cell suspension in biofilm (measurement wavelength: 600 nm)
4) A control using glucose as a saccharide was used as a control.

  The results are shown in Table 1. Here, the biofilm formation amount is shown as a ratio when the formation amount of the control is 100.

As described above, the biofilm formation promoting effect of Lactobacillus plantarum was recognized by addition of fructose.

Example 2 Promoting effect of various lactic acid bacteria on biofilm formation Sugars fructose (Wako Pure Chemical Industries), sucrose (Wako Pure Chemical Industries), and glucose (Wako Pure Chemical Industries) were used as controls.

2. Test strain
Lactobacillus brevis JCM1059 (Japan Collection of Microorganisms ), Lactobacilus gasseri JCM1131 (Japan Collection of Microorganisms), Leuconostoc mesenteroides NBRC3426 (NITE Biological Resource Center), Leuconostoc mesenteroides NBRC3832 (NITE Biological Resource Center), Pediococcus pentosaceus JCM5890 (Japan Collection of Microorganisms), Experiments were performed using Bifidobacterium bifidum NBRC100015 (NITE Biological Resource Center).

3. Experimental method 10 ml MRS liquid test tube medium (peptone (OXOID) 10.0 g, Lab-lemco Powder (OXOID) 8.0 g, Yeast extract (OXOID) 4.0 g, glucose 20.0 g, Tween80 1 ml, K ₂ HPO ₄ 2.0 g, CH ₃ COONa · 3H ₂ O 5.0 g, Citric Acid Triammonium 2.0 g, MgSO ₄ · 7H ₂ O 0.2 g, MnSO ₄ · 4H ₂ O 0.05 g (54 g / l) pH 6.2) The solution was diluted 10-fold after 24 hours at a temperature of 24 ° C. and used as a test bacterial solution.

1) Preparation of biofilm and evaluation of formation promotion effect were performed as follows.
Using a 24-well microplate (flat bottom) made of PVC (polyvinyl alcohol chloride), in a container with one cover glass (for microscopic observation) per well, 0.11 M After dispensing a liquid medium (1000 μl / well) to which sugars (glucose, fructose, sucrose) are added, add the test bacteria solution (100 μl / well), cover the container, and leave it at 35 ° C. for 24-48 hours. Incubation was performed in the place.
After removing the culture solution, the attached cells and secretions and deposits are made into biofilm, 0.1% crystal violet aqueous solution (Crystal Violet (Kanto Chemical)) is added (1000μL / well), and the biofilm is stained. did.
After removing the crystal violet aqueous solution and washing twice with tap water (1000 μl / well), the remaining biofilm adhered to the glass was quantified.
The biofilm was quantified by measuring the absorbance (595 nm) of the extract obtained by extracting crystal violet from the stained biofilm using 99.5% ethanol (1000 μL / well).

2) On the other hand, the amount of cells in the biofilm was measured as follows.
The biofilm after the culture was broken by pipetting, and the bacteria in the biofilm were suspended in the culture solution, and the absorbance (600 nm) was measured.
3) The biofilm formation amount was calculated as the biofilm formation rate as follows.
Biofilm formation rate = absorbance measurement value of biofilm dyed extract (measurement wavelength: 595 nm) / absorbance measurement value of cell suspension in biofilm (measurement wavelength: 600 nm)
4) A control using glucose as a saccharide was used as a control.

  The results are shown in Table 2. Here, the biofilm formation amount is shown as a ratio when the formation amount of the control is 100.

As described above, the addition of fructose promotes the biofilm formation of Lactobacillus genus and Bifidobacterium genus Lactobacillus, Leuconostoc genus and Pediococcus genus lactic acid cocci Admitted.

Claims (2)

  A biofilm formation promoter for one or more lactic acid bacteria selected from lactobacilli, leucostock lactic acid cocci and pediococcus lactic acid cocci containing fructose.   Lactobacillus biofilm formation promoter selected from Lactobacillus plantarum and Lactobacillus gasseri containing fructose.