HU206742B - Process for formulating microorganisma - Google Patents

Abstract

Stable prepns of non pathogenic microorganisms are based on biomasses isolated from fermentation brew contg individidual or mixed strains. To 1 pt. wt of biomass 0.4-2.0 pts wt of a carrier of large specific surface is added together with 0.1-0.4 pts wt of dried milk or dried milk constituents and/or 0.1-2.0 pts wt maumitol. The mixt is granulated while being homogenised opt dried and regranulated.

Description

The present invention relates to a process for forming microorganisms comprising filtering a fermentation broth containing any of a lactobacillus, streptococcal or penicillinic microorganism, treating the harvested biomass, and then drying and producing a stable, high germ number formulation.

It is known in the literature that microorganisms are essentially fermented for two purposes. In one case, one of their metabolites is the purpose of the fermentation, in which the cell mass present is separated from the metabolite as an excess. In the other case, they want to produce a given microbe in large numbers. In these cases, it is usually a requirement that as many microbes as possible retain their viability and retain them for as long as possible. A typical area of this latter type of use is the so-called. use of starter cultures (starter cultures). Starter crops are widely used in the food industry (dairy, brewing, meat), but are also known for agricultural purposes (eg silage) and other uses. Cultures multiply when inoculated into the appropriate material and produce a beneficial change in their metabolism.

There are four types of starter cultures that are marketed and used. The simplest solution is to use the culture together with the growth medium (fermentation broth). This method assumes that the culture will be used in the short term. In addition, it has a number of disadvantages (low germ count, incorporation of ballast in the substrate, etc.) and is therefore rarely used.

In the second case, the biomass recovered from the fermentation broth (by centrifugation, separation, etc.) is suspended in a suitable liquid medium. This solution is also not advantageous because of its large volume, difficult handling and short shelf life.

The third, most widespread method is the freeze-drying (lyophilization) of microorganisms using various preservatives and carriers.

Finally, especially in the US, frozen concentration (cryoconcentration) is also used.

These latter two procedures can be called modern. However, it has the disadvantage of the relatively expensive lyophilization and germ reduction effect, the special technology of cryoconcentration and the fact that the product should be stored at a low temperature (-20 ° C or less) and the freezing chain should not be interrupted.

German Patent Application ADD 152691, entitled "Process for Microbiological Processes", discloses a fermentation process for the production of biomass and microbiological metabolism that allows sterile fermentation in a fermentation vessel under non-sterile operating conditions by preventing the contact of microorganisms during microbiological processes. with non-sterile active substances. The invention achieves this by fermentation of the microorganisms by diluting them in an aqueous medium and encapsulating them in a microcapsule with a permeable polymer membrane. The membrane of the capsule allows the diffusion of active substances to the microorganisms, without however adversely affecting the fermentation process by undesirable microorganisms. The microcapsules are treated in gas or liquid reactors in which the optimum conditions (temperature, oxygen supply, substrate) for the fermentation process are provided. In the microcapsules, the biomass produced and the metabolites of the microorganisms accumulate, even by diffusion. From the microcapsules, the biomass or microbiological metabolite is recovered by a suitable process. Thus, this process produces biomass and microbiological metabolites in a microcapsule by a costly process.

SUMMARY OF THE INVENTION It is an object of the present invention to overcome the drawbacks of the prior art by providing a method for processing cultures of various microorganisms, while maintaining viability, and providing a stable, high germ number preparation,

The present invention is based on the discovery that direct mixing of the concentrated biomass with certain large surface additives (e.g., microcrystalline cellulose) or other auxiliary materials produces a large number of well-manageable granules which maintain the viability of the microorganism for a long time.

This recognition is also noteworthy because the vital functions of the microorganism in the wet biomass are barely limited, and thus the maintenance of a high germ count for an extended period of time is not unexpected.

The invention thus provides a process for converting non-pathogenic microorganisms (lactobacillus, streptococci, penicillium) into a stable formulation by separating the biomass from the fermentation broth containing the particular microorganism and then adding 0.42.0 wt. and 0.11.4 parts by weight of milk powder or milk powder ingredient or 0.01-0.5 parts by weight of glycerol or lecithin, optionally 0.1-2.0 parts by weight of mannitol, and granulating during homogenization.

Micro-crystalline cellulose, silicic acid, a silicon-based formulation, preferably metal silicate, lactose, casein, albumin, globulin or a mixture thereof are preferably used as a high surface carrier.

According to the present invention, a large number of Lactobacillus jensenii granules are produced by homogenizing the biomass containing the particular microorganism with 1.2-1.6 parts by weight of microcrystalline cellulose and 0.4-0.8 parts by weight of milk powder.

The same procedure is used for the preparation of Lactobacillus plantarum granules, in which the biomass containing the given microorganism is comprised of 1.2-1.5 parts by weight of microcrystalline cellulose, 0.01-0.03 parts by weight of lecithin, 1.0-1.1 parts by weight of milk powder and 15-0.20 parts by weight of mannitol.

In the process, the mixture of additives and their relative proportions may vary depending on the species and strain of microorganism processed.

HU 206 742 Β

The wet granules obtained by the process of the present invention having a dry solids content of 35-401% are placed on a tray dryer of 5-10 mm thickness and reduced to 1-5% moisture in a vacuum or air blast oven at 20-30 ° C.

The dried product is ground or granulated to a size of 50300 μτη in a suitable mill.

The material thus obtained may be further diluted, depending on the intended use, and may sometimes be applied to various carriers.

Characterization of the microorganisms used in the process of the invention:

Lactobacillus jensenii

Straight, 3-5 μτη long, non-stinging, Gram-positive rods. On the Greyhound, they form white to greyish-white colonies, and the broth is evenly clouded. Fructose, glucose, maltose are broken down, not cellobiose, gluconate, lactose, mannitol.

They grow at +15 ° C, multiply well at 2224 ° C, optimum at 30-32'C.

In non-buffered glucose soup, a final pH of 4.1 to 4.4 is set.

Streptococcus faecium

Gram-positive bacterium of the family Lactobacillaceae, genus Streptococcus. It does not form spores.

The cells are oval, approx. 1-2 pm large, grouped in diplococci. Occasionally they form a short chain, and longer chains occur in the culture medium. They are usually stationary, but there are also moving versions.

On the Greyhound approx. It forms a glossy, smooth, whitish colon with a diameter of 3-5 mm and rarely pigments.

Optional anaerobes. They are poorly cultivated on conventional media. Their temperature optimum is around 35 ° C, but they also grow at 10 and 45 ° C.

It fermentes lactose, maltose, cellobiose, fructose, galactose.

The strains also rarely reduce nitrate. Glycerol is utilized only under aerobic conditions. Adjustable pH in glucose broth is 4.0-4.4.

Lactobacillus plantarum

Straight, rounded tip, 3-8 μιη long, non-motile, rod-shaped, Gram-positive bacteria.

On the Greyhound, they form white colonies, and the broth is evenly clouded.

They produce DL-lactic acid and do not reduce nitrate.

They grow at 15 'C, grow well at 22-24' Con, optimum growth at 30-32 'C.

It breaks down glucose, fructose, maltose, cellobiose, gluconate, lactose, mannitol.

Penicillium conidium

Taxonomic location: class of conidia fungi, family Koniliaceae.

Vegetative fungi, their sexual reproduction is unknown. The culture consists of real mycelia, which are multicellular and are divided by transverse walls. The transverse walls have a soft pore.

They reproduce with conidia attached to conidia supports.

In vegetative breeding, conidia reservoirs and conidia are colorless. The conidia are formed in a chain.

In a process for forming microorganisms according to the invention, the fermentation broth containing any of the lactobacilli, streptococci or penicillium microorganisms is filtered, the biomass obtained is treated, then dried, milled, homogenized and granulated, if necessary, granulated with the biomass fraction. cellulose or silicate composition or silica, and 10-140 parts by weight of milk powder or milk powder constituents, preferably lactose, albumin or mixtures thereof, and from 1 to 50 parts by weight of glycerol or lecithin, optionally from 10 to 200 parts by weight of mannitol.

The invention is illustrated by the following examples.

Example L

Preparation of granules of Lactobacillus jensenii.

The following preservatives and excipients are added to 5 kg dry matter biomass of 5 kg Lactobacillus jensenii (MIMNG 270) obtained from 100 l fermentation broth (germ number: 2.2x10 '°):

Lactobacillus jensenii biomass 5000g lecithin lOOg milk powder (lean) 2500g lactose 2500g MCC (microcrystalline cellulose) 7500g Put these materials in a suitable kneading machine-

and granulate at 20 ° C for 20-30 minutes. The wet granulate thus obtained was spread on trays of 5-6 mm thickness, placed in a vacuum dryer and dried at 25 ° C for 30 hours. The dried material has a moisture content of 4.0 to 4.21%. The dried product is passed through a grinding machine until the granule size is reduced to 70-70 µm. The dry ground homogenized granulate (12,850 g) thus obtained is suitable for further use.

Based on substantiated tests, it was found that the germ number of the granules treated according to our method, as compared to the germ number of the untreated (control) preparation removed from the same fermentation broth, is as follows: (measurements at 25 ° C)

Date of use Plate count treated granules in untreated control material starting time 2,4x10 ° * 2,35x10 '° After 1 month 1,9x10 '° 5.8x10 ⁹ 4 months after 1.2x10 '° ⁷ 5,3xl0 6 months later 1,1x10 '° 2.8x10 ⁵ 8 months later 0,99x10 '° 7.2x10 ⁴ 10 months later 9.5x10 ⁹ 3.8x10 ⁴ 12 months after 9, lx10 ⁹ 6.4x10 ³

HU 206 742 Β

It is clear from the comparative data that the stability of the microorganism treated by the method of the invention is significantly better than that of the untreated control.

It will also be appreciated that the use of the composition treated according to the invention in use does not result in an order of magnitude reduction in use.

Example 2

Preparation of granules of Streptococcus faecium. 10

5.5 kg of Streptococcus faecium (MIMNG 174) separated from 100 l of fermentation broth (germ number: 1.2x10) are mixed with the following preservatives and excipients:

Streptococcus faecium biomass 5500g 15 glycerol 150 ml mannitol 2500g albumin 500 g lactose 2000 yrs ultrasil (fatty acid) 6000g 20 The materials are kneaded in about 25 ° C

Granulate for 20-30 minutes. The wet granules thus obtained were placed in trays of 5-6 mm thickness, placed in an air-blast oven and dried at 25 ° C for 30 hours. The dry granulate has a moisture content of 4.0-4.2% by weight. The dried material was ground to a particle size of 70-80 µm. The resulting homogenized powder (8.2x10 germ number) can be mixed with other materials as desired. During use, the reduction in germ number does not exceed one order of magnitude. 30

Example 3

Production of Lactobacillus plantarum granules.

The 1 kg biomass of Lactobacillus plantarum (MIMNG 35 269) obtained by filtration of the starting fermentation broth (germ number 4.2x10 10) is homogenized, granulated and dried with 1200 g microcrystalline cellulose, g lecithin, 1000 g milk powder and 160 g mannitol. The final weight of the dried granulate is 2100 g and the germ number is 3.9-10 °.

Example 4

Preparation of Penicillium Conidium Granules.

7 kg of Penicilium sp.

(MIMNG 1058) Biomass (conidia) with a dry matter content of 6.3% by weight are kneaded with protective and auxiliary substances as follows:

Penicillium sp. (conidium) 7000 g glycerol 75 g mannitol 2500 g

MCC (microcrystalline cellulose) 7500 g 50

The biomass is kneaded with the excipients at 20-30 ° C for about 30 minutes. The wet granules were placed in trays of 5-10 mm thickness, placed in a vacuum oven and dried at 25 ° C for 30 hours. The dry granulate has a moisture content of 3.5 to 3.6% by weight.

The dry granulate is ground to a grain size of 150-200 µm. The weight of the milled final product is 10 kg and the live germ number is 10 ', which is in the order of magnitude below the initial germ number.

Advantages of the process according to the invention:

- simple technology:

- no special equipment is required during the procedure;

- the process does not require high specific energy;

- the process provides a high germ number formulation for 6 or 12 months stability;

- this preparation can be used advantageously in the food industry (eg dairy, beer, meat industry) to improve taste, quality, shelf life and to produce feed having the above mentioned advantages in agriculture.

PATENT CLAIMS

Claims (5)

PATIENT INDIVIDUAL POINTS A method for forming microorganisms, comprising filtering a fermentation broth containing any of the lactobacillus, streptococcal or penicillium microorganisms, treating the recovered biomass, drying, grinding, homogenizing and granulating, optionally regranulating, comprising 100 parts by weight of biomass with 40 to 200 parts by weight of microcrystalline cellulose. or with a silicate composition or silica, and optionally 10 to 140 parts by weight of powdered milk or milk powder, preferably lactose, albumin, or a mixture thereof, and 1-50 parts by weight of glycerin or lecithin, optionally 10-200 parts by weight of mannitol. Process according to claim 1, characterized in that the biomass containing the microorganism Lactobacillus jensenii (MIMNG 270) is homogenized and granulated with 140-160 parts by weight of microcrystalline cellulose, 40-60 parts by weight of milk powder, 40-60 parts by weight of lactose, 1-3 parts by weight of lecithin. 3. The method of claim 1, wherein the microorganism comprising the microorganism Lactobacillus plantarum (MIMNG 269) is homogenized and granulated with 120-150 parts by weight microcrystalline cellulose, 1-3 parts by weight of lecithin, 100-110 parts by weight of milk powder and 15-20 parts by weight of mannitol. 4. The method of claim 1, wherein the microorganism comprising the microorganism of Streptococcus faecium (MIMNG 80 174) is 100-120 parts by weight of silica, 8-12 parts by weight of albumin, 1-3 parts by weight of glycerol, 40-60 parts by weight of mannitol, 3050 parts by weight \ t homogenized and granulated with lactose. 5. The method of claim 1, wherein the penicillium sp. (MIMNG 1058) The biomass containing the microorganism (MIMNG 1058) is homogenized and granulated with 100-120 parts by weight of microcrystalline cellulose, 30-40 parts by weight of mannitol, 1-2 parts by weight of glycerol. Published by the National Inventory Office, Budapest Responsible for release: dr. Gabriella Szvoboda Head of Department ARCANUM Bt.-BUDAPEST