CZ201327A3 - Strain of Lactobacillus paracasei RIBM 2-107 bacteria and its use in the preparation of no-alcoholic and low alcoholic beverages of fruit and/or grain substrates - Google Patents

Abstract

Lactobacillus paracasei strain RIBM 2-107, which fermentes glucose, sucrose, maltose, galactose, melezitose, raffinose and gluconate to form lactic acid or a mixture of lactic acid, acetic acid and ethanol, and does not ferment sorbose, mannitol, sorbitol, glycerol and N -acetylglucosamine. A method for producing non-alcoholic and low-alcoholic beverages in which a fruit and / or cereal substrate is fermented which comprises at least one sugar from the group consisting of glucose, fructose, sucrose, maltose, galactose, melezitose, raffinose, arabinose, xylose, gluconate, the fermentation being carried out by a strain of bacteria Lactobacillus paracasei RIBM 2-107, or mixtures thereof with at least one other strain.

Description

Bacterial strain Lactobacillus paracasei RIBM 2-107 and its use in the production of soft and low-alcoholic beverages from fruit and / or cereal substrates

Field of technology

The invention relates to a new strain of Lactobacillus paracasei RIBM 2107.

The invention further relates to the use of this bacterial strain in the production of non-alcoholic and low-alcoholic beverages from fruit and / or cereal substrates.

Prior art

Soft drinks are currently commonly obtained by diluting fruit or vegetable juices or syrups with drinking water. They can be modified by carbonation, pasteurization, addition of sugars and various additives (eg citric acid, caramel, natural aromas, herbal extracts, vitamins, minerals, etc.).

Alcoholic beverages, such as beer and cider, and optionally their low-alcohol variants, are then commonly obtained by fermenting fruit and / or cereal substrates via yeasts of the species Saccharomyces cerevisiae, Saccharomyces pastorianus and Saccharomyces bayanus.

The essence of the invention

The invention relates to the strain Lactobacillus paracasei RIBM 2-107, which is a gram-positive, facultatively heterofermentative mesophilic non-sporulating rod-shaped bacterial strain characterized by fermenting glucose, fructose, sucrose, maltose, galactose, melezitose, raffinose and raffinose, however, it does not ferment sorbose, mannitol, sorbitol, glycerol and Nacetylglucosamine, and according to the culture medium used and fermentation conditions it is

PS387ÓCZ • 2 'capable of producing lactic acid or a mixture of lactic acid, acetic acid and ethanol.

This strain, which occurs naturally in fermented foods, yoghurts, raw milk, fermented plant material and, for example, the human digestive tract, has been isolated from kefir grains, which are, as described, for example, in Withuhn et al .: „Characterization of the microbial population at different stages of Kefir production and Kefir grain mass cultivation“, Int. Dairy J. 15: 383-389, 2005, symbiotic cultures of lactic acid bacteria, vinegar bacteria, yeasts and fibrous fungi in proteins, 10 fats and sugars. To isolate the Lactobacillus paracasei strain RIBM 2-107, 0.1 ml of kefir grain homogenate in sterile distilled water was inoculated onto a Petri dish containing MRS (de Man, Rogosa, Sharpe) agar. To inhibit the growth of competing yeasts and fungi present in kefir grain, the antibiotic cycloheximide (also known as actidione) was added to this substrate at a concentration of 25 mg / l, and to inhibit the growth of competing gram-negative bacteria β-phenylethanol at a final concentration of 0.3 volume. %. The cultivation was then carried out anaerobically in a microbiological incubator at a temperature of 28 Ό (the most suitable temperature is about 26 to 30 Ό) for 5 to 7 days. After increasing the culture of bacteria on the surface of the agar, this culture was transferred by a sterilized inoculum loop to a new Petri dish with MRS agar and inoculated with a so-called cross-smear, in which the concentration of bacteria on the agar was diluted. This procedure was repeated until the bacterial culture grew into visibly separated colonies. Subsequent isolation of the Lactobacillus paracasei RIBM 2-107 strain was then performed by inoculating a selected colony onto a Petri dish containing 25 MRS agar.

The isolated strain was then characterized by biochemical assays and species-specific PCR.

In other variations, the procedure described in Witthuhn et al .: "Characterization of the 30 microbial population at different stages of Kefir production and Kefir grain mass cultivation", Int. Dairy J. 15: 383-389, 2005, in which MRS agar containing ethanol at a concentration of 30 g / l and an antibiotic is used as isolation medium

PS387ÓCZ cycloheximide at a concentration of 5 g / l. The petri dishes are then incubated anaerobically for 5 to 10 days at 30 ° C.

In addition, it is possible to use the procedure described in Simova et al .: "Lactic acid bacteria and yeasts in kefir grains and kefir made from them", J.

5- Industrial Microbiol. Biotechnol. 28: 1-6, 2002, in which MRS agar and Rogosa agar containing whey are used, after which the inoculated Petri dishes are incubated for 3 to 7 days at 30 Ό (for isolation of mesophilic lactobacilli) and 37 Ό (for isolation of thermophilic lactobacilli), aerobically and anaerobically in both temperature regimes.

The strain is stored in the Collection of Microorganisms of the Research Institute of Brewing and Malting, a.s. - RIBM (Research Institute of Brewing and Malting) under RIBM No 2-107.

This strain of bacteria has surprisingly advantageous properties for use in the preparation of soft and low-alcoholic beverages, as it ferments a wide range of sugars from various sources - such as fruits, cereals, or mixtures thereof in various proportions, etc., while producing a suitable amount of acids. , which lower the pH of the beverage to a value of 3.40 to 3.75, which has a beneficial effect on its final taste, without the need for further adjustment and without the formation of undesirable aromas or flavors. In addition, it also produces a small amount of 20% alcohol - depending on the culture medium used and the fermentation conditions, about 0.05 to 1% by volume.

The Lactobacillus paracasei strain RIBM 2-107 survives well in common liquid substrates (eg MRS, YPD, etc.), even in the frozen state, so it is suitable for short-term and long-term storage.

For the production of beverages, this bacterial strain is then preferably used in particular in the form of a bacterial concentrate, either alone or in a mixture with other strains, preferably, for example, with the bacterial strain Lactobacillus sp. RIBM 2-108 in any ratio.

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Examples of embodiments of the invention

Example 1: Preparation of bacterial concentrate

A solution containing at least one source of fermentable sugar (glucose and / or sucrose) and a source of proteins, vitamins and minerals required for the growth of lactic acid bacteria is used as a substrate for the multiplication of the Lactobacillus paracasei RIBM 2-107 strain. For example, glucose-containing broth, bacteriological peptone and yeast extract can be prepared. This bacterial strain is then cultured at a temperature of about 26 to 30 ° C, the culturing time being determined depending on the required final amount of bacteria, resp. according to the requirements for the fermentation of the beverage. After culturing, it is advantageous if the culture of bacteria formed is repeatedly centrifuged and washed with sterile water to remove the culture substrate, since the presence of substrate components in the beverage could adversely affect its resulting organoleptic properties.

In this way, a concentrate containing 100 million to 10 billion, or another suitable number, of living cells of the Lactobacillus paracasei strain RIBM 2-107 is prepared as needed, which can be used directly for the production of the desired beverage. In this case, either the Lactobacillus paracasei strain RIBM 2-107 or a mixture thereof with other strains is used exclusively for the fermentation of the substrate, preferably, for example, with the Lactobacillus sp. RIBM 2-108.

Example 2: Preparation of a low-alcohol apple drink using Lactobacillus paracasei strain RIBM 2-107

A low-alcohol apple beverage was prepared from 20 liters of apple cider, to which a centrifuged bacterial concentrate of Lactobacillus paracasei strain RIBM 2-107 prepared according to the procedure of Example 1 was added at a concentration of 3 g per 1 liter of must. Subsequent fermentation took place in a stainless steel barrel at 21 Ό for 7 days. After this time, the fermentation was stopped and the formed beverage was pasteurized, after which its pH, fermentation, alcohol content, concentration of diacetyl, pentanedione, acetaldehyde, lactic acid, malic acid, citric acid and acetic acid were measured. Except

PS387ÓCZ was further performed by its sensory evaluation by the tasting committee. Selected parameters of the beverage prepared in this way are listed in Table 1.

Table 1

Measured parameter Value Alcohol (% vol.) 0.62 Fermentation (%) 7.65 Extract (% wt.) 9.43 pH 3.70 Diacetyl (pg / l) 128 Pentandion (pg / l) 113 Acetaldehyde (mg / l) 7.9 Malic acid (mg / l) 991,33 Lactic acid (mg / l) 5830,256 Citric acid (mg / l) 366,161 th most common Acetic acid (mg / l) 352,529 th most common

The drink did not show any sensory defects, only in addition to the apple scent, there was also a faint lemon to grapefruit scent, which was evaluated as refreshing and pleasant. The overall subjective impression was above average.

Then this drink was bottled in bottles, part of which was stored at a temperature of 4 Ό and part at a temperature of 20 O, while its durability, respectively. its appearance 10 and taste parameters. The drink was stable even after 6 months of storage at a temperature of 20 O, when there was no grain of its color or its sensory properties.

Given that the beverage prepared in this way contained 0.62% by volume of alcohol, it is in accordance with the Decree of the Ministry of Agriculture No. 115/2011 Coll. o Low alcohol drink.

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Example 3: Preparation of a non-alcoholic grape drink using the bacterium strain Lactobacillus paracasei RIBM 2-107

The beverage was prepared as in Example 2, except that instead of apple cider, grape must mixed with a 3: 1 ratio of potable water was used. Selected parameters of the finished beverage are listed in Table 2.

Table 2

Measured parameter Value Alcohol (% vol.) 0.12 Fermented (%) 6.50 Extract (% wt.) 8.12 pH 3.75 Diacetyl (pg / l) 92 Pentandion (pg / l) 80

The drink did not show any sensory defects, it was evaluated as refreshing with a pleasant fruity aroma. The overall subjective impression was above average.

The drink was stable even after 6 months of storage at a temperature of 20 Ό, when its color and its sensory properties did not change.

Given that the beverage prepared in this way contained 0.12% by volume of alcohol, it is in accordance with the Decree of the Ministry of Agriculture No. 115/2011 Coll. for a soft drink.

Example 4: Preparation of a malt flavored non-alcoholic fruit drink using Lactobacillus paracasei strain RIBM 2-107 in combination with Lactobacillus sp. RIBM 2-108

The beverage was prepared as in Example 2, except that a mixture of apple cider and brewer's wort was used instead of apple cider.

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in a ratio of 1: 1, to which 1.5 g of a centrifuged culture of Lactobacillus paracasei RIBM 2-107 and 1.5 g of a culture of Lactobacillus sp. RIBM 2108 per 1 liter of mixture. Fermentation, filling and shelf life monitoring were performed under the same conditions as in the previous examples. Selected parameters of the finished beverage are listed in Table 3.

Table 3

Measured parameter Value Alcohol (% vol.) 0.26 Fermentation (%) 12.50 Extract (% wt.) 7.98 pH 3.28 Diacetyl (pg / l) 173 Pentandion (pg / l) 105

The drink was evaluated as refreshing, with a pleasant malty flavor. The overall subjective impression was above average.

The drink was stable even after 6 months of storage at a temperature of 20 O, when its color and its sensory properties did not change.

Given that the beverage prepared in this way contained 0.26% by volume of alcohol, it is in accordance with the Decree of the Ministry of Agriculture No. 115/2011 Coll. for a soft drink.

Depending on the requirements for the fermentation of the beverage and / or the resulting volume of alcohol and / or according to the properties and composition of the substrate used, approx.

Claims (4)

PATENT CLAIMS 1. Lactobacillus paracasei strain RIBM 2-107 which ferments glucose, fructose, sucrose, maltose, galactose, melezitose, raffinose 5 arabinose, xylose and gluconate to form lactic acid or a mixture of lactic acid, acetic acid and ethanol, and does not ferment sorbose, mannitol, sorbitol, glycerol and N-acetylglucosamine. 2. A method for producing non-alcoholic and low-alcoholic beverages by fermenting a fruit and / or cereal substrate containing at least 10 one sugar of the group glucose, fructose, sucrose, maltose, galactose, melezitose, raffinose, arabinose, xylose, gluconate, characterized in that the fermentation is carried out by a strain of Lactobacillus paracasei RIBM 2-107 according to claim 1. Method according to claim 2, characterized in that the fermentation takes place with 15 mixtures of at least two strains of bacteria, one of which is a bacterial strain Lactobacillus paracasei RIBM 2-107 according to claim 1. Process according to claim 3, characterized in that the fermentation is carried out with a mixture of a strain of Lactobacillus paracasei RIBM 2-107 according to claim 1 and a strain of Lactobacillus sp. RIBM 2-108.