DE4103755C1 - Process for stimulating growth of Bifidobacterium, etc. - comprises using substrate of oligosaccharide esp. lactose and co:substrate of fructose - Google Patents

Abstract

Process for stimulating the growth of Bifidobacteria and homofermentive bacteria forming lactic acid comprises using a substrate of oligosaccharides esp. lactose, and a cosubstrate of 0.01-0.5(0.04) g/g fructose. USE/ADVANTAGE - For forming yoghurt etc., special dietetic agents or pharmaceuticals, and esp. for re-establishing a healthy microflora in the intestines or vagina, following disturbance. To produce lactose, the microbes must first form the necessary enzymes, causing a lag phase in the culture. This is shortened by adding a co-substrate. The usual co-substrate, glucose, encourages a hetero-fermentation with prodn. of CO2, but fructose encourages only homo-fermentation.

Description

The invention relates to a method for stimulation growth of bifidobacteria and homofermentative Lactic acid-forming bacteria (hereinafter homofermentative Called lactic acid bacteria).

Lactic acid-forming, Gram-positive bacteria are both in the food industry (production of Yoghurt etc.) as well as for dietary products or for special drugs needed. The latter will happen everything for the rehabilitation of a disturbed intestinal or Vaginal microflora used. So it is well known that just representatives of the genera Lactobacillus and Bifidobacterium special share of the physiological and symbiontic colonization of the mucous membranes internal surfaces in the body of humans and animals to have.

For the production of sour milk, yoghurt etc. available the food industry over decades tested Cultivation process in milk, suggesting many food technologically interesting types of bacteria or equally applicable to these related bacteria is.

Somewhat more complicated is the cultivation of microorganisms, those without special growth additives in milk can not be cultivated, only sparse to development  come, or even strict exclusion of atmospheric oxygen like that for most in the Micro-organisms occurring bacteria, the anaerobes, applies.

Of particular interest here are the species from the form rich Acidophilus group, about Lactobacillus gasseri, L. crispatus and L. acidophilus (sensu stricto) although they are not strict anaerobes as well as the much sought after bifidobacteria.

A disadvantage of these cultured for special drugs Bacteria affects the required significant Effort out. So need the complex compound Nutrient media a rich source for amino acids and oligopeptides, furthermore vitamins and salts and a fermentable carbohydrate, such as Glucose or lactose as C source (carbon Source).

Frequently, special requirements are appropriate to the purpose additionally provided. So z. B. for Rehabilitation of a bacterial incorrect settlement on Mucous membranes bacteria not possible on glucose, but cultured on lactose as the C source, though Glucose usually represents the better substrate.  

One intends thereby a special adaptation of the Bacteria to growth on lactose after application on added lactose as soon as possible Development and thus a selective one Development advantage over the unmatched To gain germs or pathogens in the spillover whereby the latter are eliminated faster can.

In general, for the growth of cultures on lactose - cultures that make good use of lactose to be preserved - longer incubation periods accepted because, in the cell, the required Lactose degrading enzymes first induced and synthesized Need to become. This involves complicated cellular regulatory mechanisms. This induction phase prevents a rapid growth of crops, one speaks of a pronounced lag phase in the Growth curve.

It is also common to find that in such a In fact, even in a lagging phase, the cultures even Energy crisis advised, so that the actual start of propagation, the cell division, over hours on itself wait to compare growth to glucose. The electron balance of the fermentation equation is shifted unfavorably, and so occur more difficult to assess influences in the culture conditions on. The process duration is then not subject  more only biologically based fluctuations and raises economic problems.

Therefore, to facilitate the induction process often a small amount of glucose as cosubstrate attached, on the order of 0.1% (w / v), while lactose is 1.0% in the medium. In heterofermentative lactic acid bacteria, eg. B. L. brevis or Leuconostoc species, instead became with an addition of 0.1% fructose a better Growth achieved. However, fructose is used here only as electron and hydrogen Acceptor and is converted to mannitol, causing the Regeneration of intracellular NADH (nicotinamide adenine dinucleotide, reduced) and the internal Redox balance is compensated faster.

Unfortunately, this mechanism does not occur in bifidobacteria and in homofermentative lactic acid-forming Bacteria, but only in heterofermentative Lactic acid forming bacteria. The former form in contrast to the heterofermentative lactic acid forming Bacteria (CO₂-producing bacteria) from fructose no mannitol, but use fructose, though anyway, just like a normal C source, like Lactose, glucose or other carbohydrates too.

The object of the present invention was therefore to a method of stimulating growth homofermentative lactic acid bacteria or bifidobacteria to accomplish.  

This task was surprisingly solved by that the growth of such bacteria on Lactose or other oligosaccharides with one extremely low addition of fructose stimulated.

The invention thus relates to a method to stimulate the growth of bifidobacteria and homofermentative lactic acid-forming bacteria, which is characterized by the fact that the Substrate serving oligosaccharides, in particular Lactose, 0.01 to 0.5% (w / w) fructose as cosubstrate added. By the addition of fructose is a marked shortening of the lag phase of such Cultures, as in strains of the species L. gasseri or B. Longum was found reached. So the time gain is cultivated at night Stir at least two hours with the same Inoculum. In more detailed experiments, it was shown that in addition to the shortening of the lag period no shortening of the subsequent cell division time or increasing the cell yield occurs. This physiological mechanism is not unique clarified.

It is conceivable that fructose can be called "precursor" Substrate for N-acetylglucosamine and thus the cell wall biosynthesis serves. Actually such N-acetylglucosamine derivatives of some Strains of Bifidobacteria as a "Bifidus Factor" used for cell wall synthesis.  

The effect of growth stimulation according to the invention through the use of fructose occurs during replacement the fructose is not due to glucose. According to the invention thus become the cultural activities of Bifidobacteria and / or homofermentative lactic acid forming bacteria only by the cosubstrate fructose stimulated.

In a preferred embodiment of the invention Procedure will be the lactose or the other Oligosaccharides 0.04% (w / w) fructose as cosubstrate added to the growth stimulation.

Preference is given to the inventively prepared Fermentative active microorganisms used for Production of remedies against disorders of the physiological Mucous membrane milieu, in particular of the physiological Vaginal milieus as well as against disorders of the physiological intestinal microflora.

The invention will be closer to the following examples explains:  

Example 1

Lactobacillus gasseri VPI 11 089 (ATCC 9857) is described in cultured (grams per liter of medium): Meat peptone, 10; Yeast extract, 1; Sodium acetate, 3; Sodium chloride, 5; Magnesium sulfate, 0.2; Dipotassium phosphate, 0.78; Tween 80, 0.25; Lactose (DAB 9), 20. The substances be in water (purified water DAB 9) dissolved and adjusted to a pH around 6.5. The addition of lactose as indicated takes place only after autoclaving (121 ° C for 20 min) of the lactose-free Basal medium, the lactose at about 90 ° C than sterile filtered, concentrated solution added becomes; Final concentration: 2% lactose (w / w).

Culture conditions:

Cultivation is batch-wise; as inoculum for 1 l main culture serve 4 ml preculture, cultured in MRS broth with glucose as C source (Carbon source). Pre- and main culture are each resting overnight at 37 ° C until stationary Growth phase incubated. The growth will by acidification with a calibrated pH electrode tracked; in the beginning the pH is at 6.5 and in the stationary phase at pH 4.0 to 4.2.  

Additional checks include checking for microbiological purity and identity of the strain or the culture as well as the determination of the living germ counts (CFU / ml).

As comparison medium with 0.04% fructose (DAB 9) as Cosubstrat the same medium is used; in the Addition of lactose under sterile filtration will also together with this a solution of D-fructose (DAB 9) added; Final concentration: 0.04% fructose (G / G).

The comparison of 10 culture batches with or without cosubstrate fructose shows (see following tables), that the final pH of 4.2 when cultivated with lactose alone in 21 hours incubation time is barely reached (early stationary phase) and also not a maximum germ count of about 5 × 10⁸ cfu / ml on average at 10 batches. Only in the presence of fructose as cosubstrate becomes the stationary phase, a pH at about 4.1 and germ counts by 5 × 10⁸ CFU / ml. The incubation period is also on average 19 hours considerably shorter than with lactose alone.  

Table 1

Cultivation approaches with L. gasseri VPI 11,089 in the medium with 2% lactose

Table 2

Cultivation approaches with L. gasseri VPI 11 089 in the medium with 2% lactose and 0.04% fructose

example 2

L. gasseri VPI 11 089 was used in parallel culture approaches such as cultivated in Example 1:

Approach a: Culture only with 2% lactose.
Approach b: culture batch with 2% lactose and 0.04% fructose.
Approach c: culture batch with 2% lactose and with addition of 0.04% fructose after incubation for only 20 hours.

Table 3 shows that stimulation of growth by Fructose is possible only at the beginning of culture while a later addition - if the growth on lactose already in Gear has come - has no effect.

Table 3

Cultivation of L. gasseri VPI 11,089 to 2% lactose with variation of the addition of 0.04% fructose: a) without fructose; b) with fructose; c) with later addition of fructose (after 20 hours)

example 3

L. gasseri VPI 11,089 is prepared as in Example 1 without fructose Additive cultivated. Upon reaching the early stationary phase The culture is connected to a microfiltration system and the cells freed from the medium and at the same time around the Factor 50 concentrated and washed with water. The cells be in a protective and auxiliary solution of lactose and Gelatin suspended and gently preserving viability (CFU / g) freeze-dried in a suitable plant.

The resulting amorphous material is in the ratio 1: 3 homogenized with lactose (DAB 9) in a granular mixture and 200 mg in hard gelatin capsules, Size 1, bottled; the filling consists of about 190 mg Lactose. The germ count is 2 × 10⁹ cfu / capsule.

The protective and auxiliary solution added before freeze-drying is composed as follows: lactose (DAB 9), 4.8 g; Gelatin (DAB 9), 0.3 g. These substances are in 9.2 g of water (purified water, DAB 9) dissolved with heating, bactericidally treated at 100 ° C for 30 min further processing cooled to 35 ° C.

To test the influence of fructose, capsules are also used whose filling additionally contains 6 mg of D-fructose (DAB 9).

Capsules with or without fructose are then used to compare Acidification rate after in vitro vitalization of the Germs tested on a suitable nutrient medium. For detection acidification, the nutrient medium contains a pH indicator, Water blue or China blue, the acidity of colorless after  turn blue.

The blood agar medium proved to be particularly suitable for this purpose after HACKENTHAL-BIERKOWSKI in the following modification (G / V):

Blood Agar Base # 2, | 4.0% Glucose, 1.0% China Blue, 0.03% defibrinated sheep's blood, 7.0%

After dissolving the base substrate in water becomes a pH of 7.2 adjusted with dilute sodium hydroxide solution, autoclaved and the other ingredients as sterile or sterile filtered Solutions added at about 50 ° C and after distribution in plates (Koch's plate casting) overnight at 37 ° C the excess "Crushed water" away.

To check and compare the acidification rate each one capsule with or without fructose on one half of the bright red medium and continued at 37 ° C incubated.

After just a few minutes, the heat and humidity of the Nutrient soil visible on the capsule, because this is soft, opens quickly, and the content spreads out the capsule remains. The capsule with fructose is already inside the first hour a darkening of the indicator to recognize in the culture medium; the acid formation is thus rapid on. In the control without fructose is for hours no Reaction of the indicator detectable. Only after hatching both capsules have run down and surrounded by dark colored courtyards in the medium. However, that is in the presence of fructose the farm is almost 50% larger than at the control. This shows that the physiological activity  the bacteria is significantly stimulated by fructose.

example 4

A strain of the species Bifidobacterium longum is analogous to Example 1 cultivated. The medium is slightly different. It contains no yeast extract and no Tween 80, instead but 0.017% cysteine hydrochloride for anaerobic Culture conditions.

The preculture in MRS broth (see Example 1) additionally contains 0.05% cysteine hydrochloride.

The incubation is carried out as in Example 1 by resting Night at 39 ° C. The main culture is inoculated with 4 ml per Liter of culture medium as in Example 1. The anaerobic conditions are solely by the addition of cysteine hydrochloride held.

As in Example 1, the growth is based on acid formation tracked. At the end of the growth, the pH is 4.2.

Analogously to Example 1 sets with 0.04% D-fructose (DAB 9) the Cell division earlier, so that here, too, the stationary Phase is reached earlier than in the medium without fructose. Under the latter conditions - it can be determined - varies the delay of the growth of bifidobacteria significantly or it stays out every now and then.

Claims (2)

1. A method for stimulating the growth of bifidobacteria and homofermentative lactic acid-forming bacteria, characterized in that the substrate serving as oligosaccharides, in particular lactose, 0.01 to 0.5% (w / w) fructose added as cosubstrate. 2. The method according to claim 1, characterized that 0.04% (w / w) fructose is added as cosubstrate.