GROUP OF MICROORGANISMS COMPOSED BY LACTOBACILLUS SP STRAIN K03D08, BACILLUS SP STRAIN K03B01 AND KAZACHSTANIA SP STRAIN K03K02G AND ITS COMPOSITIONS; A PROCESS FOR OBTAINING CASEIN-FREE DAIRY DERIVATIVE CONTAINING SHORT-CHAIN FATTY ACIDS AND HYDROXYLATED SHORT-CHAIN FATTY ACIDS GENERATED BY THE METABOLISM OF THE GROUP OF MICROORGANISMS

Abstract

A group of microorganisms, composed of Lactobacillus sp. strain K03D08, Bacillus sp. strain K03B01 and Kazachstania sp. strain K03K02G, which allows the hydrolysis of casein in different dairy products and the production of short-chain fatty acids and hydroxylated short-chain fatty acids; a process for obtaining a casein-free dairy derivative and enriched in short-chain fatty acids and hydroxylated short-chain fatty acids using the aforementioned microorganisms; compositions containing the microorganisms; and the production method of each of the microorganisms.

Claims

1. A group of microorganisms comprising: a) a strain of Bacillus sp. K03B01, deposited in the Chilean Collection of Microbial Genetic Resources (CChRGM) with access number 2934; b) a strain of Lactobacillus sp. K03D08, deposited in the Chilean Collection of Microbial Genetic Resources (CChRGM) with access number RGM2950; and c) a strain of Kazachstania sp. K03K02G, deposited in the Chilean Collection of Microbial Genetic Resources (CChRGM) with access number 2935.

2. A composition for producing a dairy derivative with a casein content of less than 0.5% w/v and containing short-chain fatty acids and hydroxylated short-chain fatty acids comprising: a) a first composition comprising the Bacillus sp. K03B01 strain according to claim 1 and excipients acceptable in the food industry; b) a second composition comprising the Lactobacillus sp. K03D08 strain according to claim 1; and the Kazachstania sp. K03K02G strain according to claim 1 and excipients acceptable in the food industry.

3. A method of producing the composition of microorganisms according to claim 2 comprising: a) cultivating the Bacillus sp. K03B01 strain between 12 and 96 h, with agitation between 5 and 300 rpm and a temperature between 20° C. and 37° C. in a culture medium containing skim milk; b) cultivating the Lactobacillus sp. K03D08 between 12 and 96 h, with agitation between 5 and 300 rpm and a temperature between 20° C. and 37° C. in a suitable microbiological culture medium; c) cultivating the Kazachstania sp. K03K02G between 12 and 96 h, with agitation between 5 and 300 rpm and a temperature between 20° C. and 37° C. in a suitable microbiological culture medium; d) carrying out centrifuge of each culture of microorganisms between 1,000 and 4,000 rpm for 5-30 min at 4-25° C.; e) discarding the supernatant and suspend the microbial cells in the same volume of physiological serum (NaCl 0.9% w/v); f) quantifying the optical density of the microbial cell suspensions at a wavelength of 600 nm; g) preparing a first composition comprising the Bacillus sp. K03B01 strain; and h) preparing a second composition comprising the Lactobacillus sp. K03D08 strain and comprising the Kazachstania sp. K03K02G strain in a 1:1 ratio, respectively.

4. The method of producing according to claim 3, wherein the culture medium of the first composition has the following composition: 0.25% w/v yeast extract; 0.1% w/v glucose; 1% w/v skim milk; and final pH 6.0-7.0.

5. The method of producing according to claim 3, wherein the culture medium of the second composition comprises: 1% w/v peptone, 0.8% w/v meat extract, 0.4% w/v yeast extract, 2% w/v glucose, 0.2% w/v dipotassium hydrogenated phosphate, 0.5% w/v sodium acetate trihydrate, 0.2% w/v triammonium citrate, 0.02% w/v magnesium sulphate heptahydrate, 0.005% w/v manganese sulphate tetrahydrate, 0.1% w/v tween 80, pH 6.2-6.5.

6. A method of producing a dairy product with a low casein content and containing short-chain fatty acids and hydroxylated short-chain fatty acids comprising: i. incubating the Bacillus sp. K03B01 strain in milk of bovine origin between 6 and 120 hours and at a temperature between 20° C. and 37° C., in a proportion between 0.1-10% w/v or 0.1-10% w/w, with agitation between 5-300 rpm; ii. preparing a composition comprising the microorganisms Lactobacillus sp. K03D08 and Kazachstania sp. K03K02G in a (1:1) ratio; iii. adding the composition obtained in point ii) to the solution obtained in point i) and incubating for between 12 and 120 hours and at a temperature of 20° C. and 37° C., with aeration through stirring between 0-50 rpm; iv. centrifugating the solution obtained in point iii), between 4,000 and 10,000 rpm and recovering the supernatant; v. filtering the supernatant through a 0.2-0.45 μm pore diameter membrane; vi. recovering the filtrate; and vii. lyophilizing the filtrate obtained in point vi) until total dehydration at −80° C. and between 666.6 Pa to 1999.8 Pa (5-15 mTorr).

Description

BRIEF DESCRIPTION OF FIGURES

[0010] FIG. 1. Microbial growth in solid culture medium. A: Bacillus sp. strain K03B01 growth on skim milk agar. B: Lactobacillus sp. Strain K03D08 growth on agar MRS. C: Kazachstania sp. strain K03K02G growth on agar Sabouraud.

[0011] FIG. 2. Gram stain of selected microbial strains. A: Bacillus sp. strain K03B01. B: Lactobacillus sp. strain K03D08. C: Kazachstania sp. strain K03K02G.

[0012] FIG. 3. Casein hydrolysis of selected strains. Evaluation of the hydrolysis of casein on skim milk agar Bacillus sp. strain K03B01 (A), Lactobacillus sp. strain K03D08 (B) and Kazachstania sp. strain K03K02G (C). Casein hydrolysis is visualized as a translucent halo around the inoculation well.

[0013] FIG. 4. Casein hydrolysis in casein-free dairy derivative. Acrylamide gel protein electrophoresis of proteins present in skim milk (SM) and in the samples of the dairy derivative of the present invention (DD), visualized with staining with silver salts (A) and Coomassie blue staining (B).

DETAILED DESCRIPTION OF THE INVENTION

[0014] The present invention corresponds to a group of microorganisms that allow the hydrolysis of casein in different dairy products and the production of short-chain fatty acids and hydroxylated short-chain fatty acids; a process of obtaining a casein-free dairy derivative and enriched in short-chain fatty acids and hydroxylated short-chain fatty acids using the aforementioned microorganisms; compositions containing the microorganisms; and finally to the production method of each of the microorganisms of the invention.

[0015] The present invention corresponds to a group of three microorganisms that allow the degradation of casein present in dairy products and the production of short-chain fatty acids and hydroxylated short-chain fatty acids. In a specific embodiment, the process comprises at least one Bacillus sp. isolated strain, at least one Lactobacillus sp. isolated strain, and at least one Kazachstania sp. isolated strain.

[0016] The strains were identified using conserved markers. In the case of the fungal strain, ITS4-ITS5 was used, whereas in the case of bacteria, 16S rDNA sequences were used.

[0017] All the strains described in the present invention have been deposited in the Chilean Collection of Microbial Genetic Resources (CChRGM) in accordance with the Budapest Treaty. Access numbers for each strain are specified below.

TABLE-US-00001 Microorganism Access No. Deposit date Lactobacillus sp. strain K03D08 RGM2950 Jan. 30, 2020 Bacillus sp. strain K03B01 RGM2934 Jan. 22, 2020 Kazachstania sp. strain K03K02G RGM2935 Jan. 22, 2020

[0018] In a second aspect of the invention, the composition containing the group of microorganisms of the present invention is a wettable powder composition, a concentrated suspension, liquid or paste or gel, or other systems such as encapsulation of microorganisms, such as microcapsules, nanoparticles formed by inorganic materials, such as silicon dioxide, silver or others, or organic materials, such as suitable gelling agents, such as in biodegradable polymer matrices such as agar agar, carrageenan, gelatin, among others, and excipients acceptable in the food industry.

[0019] The wettable powder and/or liquid composition of the present invention comprises at least one isolated strain selected from an isolated strain of the genus Bacillus, a Lactobacillus isolated strain and at least a Kazachstania isolated strain in a (2:1:1) ratio.

[0020] The excipients for the case of a wettable powder composition are selected from: cereal starches, lactose, talc, maltodextrin, diatomaceous earth, or a microbiologically acceptable carrier.

[0021] In the composition of the present invention, the isolated strains of Lactobacillus sp. K03D08 and Kazachstania sp. K03K02G, are present in a ratio that can be 1:1, 1:2, 2:1, 1:3, 3:1, 2:3, 3:2, 1:4, 4:1, 3:4, 4:3, 1:5, 5:1, 1:6, 6:1, 1:7, 7:1, 1:8, 8:1, 1:9, 9:1, 1:10; 10:1, 5:6, 6:5, 6:7, 7:6, 7:8, 8:7, 8:9, 9:8, with an ideal ratio of 1:1 For the formulation, the microorganisms come from pure cultures and grown in ideal liquid culture media for each of the strains indicated in this invention.

[0022] The final number of microorganisms in the composition of the present invention is: the isolated strain of Lactobacillus sp. K03D08 present between 10.sup.1 and 10.sup.15 CFU/ml or CFU/g, the isolated strain of Kazachstania sp. K03K02G is present between 10.sup.1 and 10.sup.15 CFU/ml or CFU/g, and the isolated strain of Bacillus sp. K03B01 is present between 10.sup.1 and 10.sup.15 CFU/ml or CFU/g.

[0023] Furthermore, the present invention discloses a procedure for obtaining each of the microorganisms, and this procedure involves the following steps:

[0024] 1) Cultivating the Lactobacillus sp. K03D08 strain for 12-96 h, with an agitation of 5-300 rpm and 20-37° C., in Man, Rogosa and Sharpe medium (MRS: 1% w/v peptone, 0.8% w/v meat extract, 0.4% w/v yeast extract, 2% w/v glucose, 0.2% w/v dipotassium hydrogenated phosphate, 0.5% w/v sodium acetate trihydrate, 0.2% w/v triammonium citrate, 0.02% w/v magnesium sulphate heptahydrate, 0,005% w/v manganese sulphate tetrahydrate, 0.1% w/v tween 80, final pH 6.2-6.5).

[0025] 2) Cultivating the Bacillus sp. K03B01 strain in skim milk medium (0.25% w/v yeast extract, 0.1% w/v glucose, 0.1% w/v skim milk, final pH 6.0-7.0), for 12-96 h, at 5-300 rpm and 20-37° C.

[0026] 3) Cultivating the Kazachstania sp. K03K02G strain in skim milk medium (0.25% w/v yeast extract, 0.1% w/v glucose, 0.1% skim milk, final pH 6.0-7.0), for 12-96 h, at 5-300 rpm and 20-37° C.

[0027] 4) Centrifuge each culture of microorganisms between 1.000 and 4.000 rpm for 5-30 min at 4-25° C.

[0028] 5) Discard the supernatant and suspend the microbial cells in the same volume of physiological serum (NaCl 0.9% w/v).

[0029] 6) Quantify the optical density of the suspensions obtained in point 5) at a wavelength of 595-600 nm (O.D: 600 nm).

[0030] The present invention further discloses a process for the production of a dairy derivative with a low casein content that comprises the following steps:

[0031] a) Incubating the Bacillus sp. K03B01 strain in milk of bovine origin between 6 and 120 hours and at a temperature between 20° C. and 37° C., in a proportion between 0.1-10% w/v or 0.1-10% w/w, with agitation between 5-300 rpm.

[0032] b) Preparing a composition comprising the microorganisms Lactobacillus sp. K03D08 and Kazachstania sp. K03K02G in a (1:1) ratio.

[0033] c) Adding the composition obtained in point b) to the solution obtained in point a) and incubating for between 12 and 120 hours and at a temperature of 20-37° C., with aeration through stirring between 0-50 rpm.

[0034] d) Centrifugating the solution obtained in point c), between 4.000 and 10.000 rpm and recovering the supernatant.

[0035] e) Filtering the supernatant through a membrane of 1-1000 μm pore diameter.

[0036] f) Filtering the supernatant through a 0.2-0.45 μm pore diameter membrane.

[0037] g) Recovering the filtrate

[0038] h) Lyophilizing the filtrate obtained in point f) until total dehydration at −80° C. and between 666.6 Pa to 1999.8 Pa (5-15 mTorr).

[0039] In a more specific embodiment, the incubation period of milk of bovine origin with Bacillus sp. K03B01 is between 12 and 96 hours.

[0040] In a more specific preferred embodiment, the incubation temperature of Bacillus sp. K03B01 with milk of bovine origin is between 26° C. and 37° C.

[0041] In the context of the present invention, it is understood as part of the knowledge of an expert the preparation and cultivation methods of the isolated strains. Representative methods are shown in the examples that follow hereinafter and are intended to illustrate the present invention, but not to limit its scope.

EXAMPLES

Example 1: Obtaining and Selection of Microorganisms Capable of Reducing Casein and Lactose Present in Dairy Products

[0042] The strains presented in the present invention were obtained from fermented dairy products of the probiotic type and belonging to the Center for Micro-Bioinnovation (University of Valparaiso, Valparaiso, Chile). These fermented products were previously characterized through microbiological and metagenomics studies, identifying genes and enzymes related to casein and lactose hydrolysis, in addition to the production of short-chain fatty acids. In this sense, it was considered that the fermented products are natural sources of microorganisms, genes, enzymes and biomolecules of applied interest, and therefore the source or origin of the strains of the present invention.

[0043] With the aim of isolating the microorganisms present in these dairy fermentations, serial dilutions were performed in physiological serum and seeded on skim milk agar supplemented with yeast extract and casamino acids. From these microbial cultures, the isolation and selection of colonies grown at a temperature of 30° C. was performed. With the colonies we proceeded to generate a bank of microorganisms. To determine the properties and activities of interest, each of the isolated strains was grown in the following selective culture media (Table 1).

TABLE-US-00002 TABLE 1 Culture media used and relevant phenotypes Culture medium Relevant phenotype Skim milk agar Determination of hydrolysis of casein MRS agar Identification of bacteria that produce lactic acid GYC agar Identification of acetic acid-producing microorganisms Sabouraud agar Yeast isolation

[0044] The isolated strains were microbiologically characterized (gram stain, morphology) (FIG. 2, Table 2), molecularly (Table 3) and biochemically (Table 4). Additionally, the ability of these microbial strains to hydrolyze casein present in milk was determined (Table 5). From this process, strains of different microorganisms with phenotypes of interest such as lactose metabolism, glucose fermentation and protein hydrolysis were selected.

TABLE-US-00003 TABLE 2 Characterization of the selected strains and relevant phenotypes Strain Characteristics K03B01 Bacteria. Gram-positive bacillus. Casein hydrolysis. K03D08 Bacteria. Gram-positive bacillus. Beta- galactosidase activity, lactose metabolism. Lactic acid production. K03K02G Yeast. Acetic acid production

TABLE-US-00004 TABLE 3 Molecular characterization of the selected strains, by means of amplification and sequencing of 16S rDNA for strains K03B01 and K03D08 while for strain K03K02G the ITS4-ITS5 region was used. Strain Molecular identification Homology K03B01 Bacillus sp. 100% Bacillus aryabhattai K03D08 Lactobacillus sp. 100% Lactobacillus plantarum K03K02G Kazachstania sp. 100% Kazachstania unispora

TABLE-US-00005 TABLE 4 Enzymatic activity of the selected strains Reaction/ Bacillus sp. Lactobacillus sp Kazachstania sp. Proof Enzymes K03B01 K03D08 K03K02G ONPG β-galacto- + + + sidase VP Acetoin + + + GEL Gelatinase + − −

TABLE-US-00006 TABLE 5 Casein hydrolysis in selected strains Hydrolysis halo in skim Casein Strain milk agar (mm) Quantification* Bacillus sp. K03B01 60 <0.00025 g/100 ml   Lactobacillus sp. K03D08 15 >2.5 g/100 ml Kazachstania sp. K03K02G 0 >2.5 g/100 ml *Casein quantification in skim milk culture inoculated and incubated with the selected strains.

Example 2: Optimization of the Production Process of a Casein-Free Dairy Derivative Enriched in Short-Chain Fatty Acids and Hydroxylated Short-Chain Fatty Acids

[0045] During this development, three production processes were evaluated to obtain a dairy product, casein-free, low in lactose and enriched in short-chain fatty acids and hydroxylated short-chain fatty acids. Under these conditions, the microorganisms to be used in the process and its physicochemical conditions were evaluated.

TABLE-US-00007 TABLE 6 Production conditions evaluated for the dairy derivative Condition 1 Condition 2 Condition 3 Microorganisms Bacillus sp. strain Bacillus sp. strain Bacillus sp. strain K03B01 K03B01:Lactobacillus sp. K03B01 Lactobacillus sp. strain K03D08:Kazachstania strain K03D08:Kazachstania sp. strain K03K02G (2:1:1). sp. strain K03K02G (1:1). Process type 1 Step 1 Step 2 Steps Process Inoculate the Inoculate the microbial Step 1 microorganism in skim composition in skim Inoculate the Bacillus sp. milk in a proportion milk in a proportion K03B01 strain in skim milk of 2% w/v. of 2% w/v. in a proportion of 2% w/v. Incubate at 37° C. Incubate at 37° C. Incubate at 30° C. for 48 h, for 48 h, with agitation. for 48 h, without agitation. at 150 rpm. Centrifuge at 6000 RPM Centrifuge at 6000 RPM Step 2 for 30 min at 4° C. for 30 min at 4° C. Inoculate the resulting Filter the supernatant Filter the supernatant product of Step 1 with the through a membrane through a membrane microbial composition with a 0.22 μm pore with a 0.22 μm pore comprising the strains diameter. diameter. Lactobacillus sp. K03D08 and Kazachstania sp. K03K02G (1:1) in a proportion of 2% w/v. Incubate at 37° C., without agitation for 48 h. Centrifuge at 6000 RPM for 30 min at 4° C. Filter the supernatant through a membrane with a 0.22 μm pore diameter. Product Casein: <0.00025 g/100 ml Casein: <0.00025 g/100 ml Casein: <0.00025 mg/100 ml characteristics Lactic acid: N.D. Lactic acid: N.D. Lactic acid: 1.642 g/100 ml Acetic acid: N.D. Acetic acid: N.D. Acetic acid: 0.402 g/100 ml Lactose: 4.2 g/100 ml Lactose: 4.2 g/100 ml Lactose: <3 g/100 ml N.D.: Not detected

[0046] Based on the conditions evaluated, the Condition 3 was selected as a production process of a casein-free dairy derivative that contains short-chain fatty acids and hydroxylated short-chain fatty acids.

Example 3: Comparative Example Between Formulations with Different Compositions of Microorganisms

[0047] Given that the individual analysis of the strains allowed to establish that the microorganism Bacillus sp. K03B01 strain had the highest casein hydrolysis capacity, this microorganism was selected for the process, while the microorganisms Lactobacillus sp. strain K03D08 and Kazachstania sp. strain K03K02G were selected for their ability to metabolize lactose and glucose and produce short-chain fatty acids and hydroxylated short-chain fatty acids. The characterization of the products obtained in each of the combinations of microorganisms and the different production processes are detailed below.

TABLE-US-00008 TABLE 7 Characterization of the dairy derivative generated through the different production processes Bacillus sp. strain Bacillus sp. strain Bacillus K03B01:Lactobacillussp. K03B01 Lactobacillus sp. sp. strain strain K03D08:Kazachstania strain K03D08:Kazachstania Microorganisms K03B01 sp. strain K03K02G (2:1:1). sp. strain K03K02G (1:1). Process type 1 Step 1 Step 2 Steps Casein <0.00025 g/100 ml <0.00025 g/100 ml <0.00025 g/100 ml    Acetic acid ND ND 0.402 g/100 ml Lactic acid ND ND 1.642 g/100 ml Lactose     4.2 g/100 ml     4.2 g/100 ml  .sup. <3 g/100 ml ND: Not detected

TABLE-US-00009 TABLE 8 Characterization of the dairy derivative obtained in the present invention and of the skim milk used as a substrate for the production process Skim milk Dairy Characteristic (substrate) derivative Casein >2.5 g/100 ml < 0.00025 g/100 ml  .sup.  Acetic acid ND 0.402 g/100 ml Lactic acid 0.08 g/100 ml 1.642 g/100 ml Lactose  4.6 g/100 ml  .sup. <3 g/100 ml N.D.: Not detected