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

[0001] This Application is a 371 of PCT/IB2020/051608 filed on Feb. 25, 2020, the contents is incorporated herein by reference.

[0002] The present invention corresponds to a group of microorganisms that allow the hydrolysis of casein in different dairy products, compositions containing the microorganisms, production method of each of the microorganisms of the invention and a process for obtaining a casein-free dairy derivative containing short-chain fatty acids and hydroxylated short-chain fatty acids.

[0003] Dairy derivatives with reduced casein content are products that have increased their consumption because many of them have been reported as less allergenic compared to other types of products, making them potentially useful for example for newborn food formulas. Casein hydrolysates can be traditionally produced using proteolytic enzymes, which are responsible for hydrolyzing casein (the main milk protein). However, using enzymes in different production processes makes them more expensive since the enzymes themselves are the more expensive inputs in various production processes. Additionally, the products that have a low casein content can be produced through thermal treatments or acid or alkaline hydrolysis, which can produce severe degradation in several of the amino acids present in milk, reducing its nutritional value. This loss in its nutritional value generates additional costs in the production process since the resulting dairy product must be supplemented with all the nutrients that were lost during the casein hydrolysis stage. Furthermore, current production processes are carried out between 45° C. and 60° C., generating extra costs to be able to maintain said temperature range during the production time period (between 36 to 56 hours).

[0004] Different attempts have been made in search of finding solutions to reduce costs associated with this production process, within which the use of microorganisms has taken a relevant importance.

[0005] Microorganisms stand out for being life forms that have a wide metabolic diversity, which, for example, translates into the presence of extracellular hydrolytic enzymes (secreted or membrane-anchored), capable of degrading the different substrates available in their environment so that they can be metabolized as monomeric units. Among the enzymes of interest are the proteases since these have been used in different industrial applications.

[0006] Specifically, regarding the degradation of milk proteins, publications of patent documents have been described that seek, in most cases, to degrade casein by enzymatic methods, for example document US2017037442A1 describes a casein hydrolysate through the controlled hydrolysis of a substrate rich in this protein, using a proteolytic preparation derived from a fungus of the genus Aspergillus. It is described that the produced hydrolysate has at least a 98% reduction in terms of its antigenicity and regarding the sizes of the peptides generated, it is indicated that these, in general, are greater than 5 kDa. It is indicated that the casein-rich raw material may be cow's milk. The document WO2016066758A1 describes hydrolysates and their use in the treatment and/or prevention of diarrhea, since these hydrolysates inhibit the enzyme neprilysin which is associated with this pathology. The method for obtaining these hydrolysates involves the incubation of the milk to be treated with preparations of proteases selected from the group of neutral proteases, alkaline proteases, thermolysins and mixtures thereof. The document US2011097760A1 describes a method of producing a casein hydrolysate using an endopeptidase. This document explicitly describes what the amino acid composition of said enzyme would be. The document U.S. Pat. No. 5,486,461A describes an enzymatic method for obtaining casein hydrolysates that uses three different proteolytic enzymes. It is further noted that this method allows to obtain a preparation that completely hydrolyzes casein. However, due to the associated costs involved in the use of pure enzyme preparations, the disclosure of documents describing the degradation of casein by means of the use of microorganisms has been increasing.

[0007] The document US2009214498A1 generally describes a method of producing antimicrobial peptides by incubating a biologically pure culture of Lactobacillus acidophilus, strain DPC6026, using milk or a product derived from milk as raw material.

[0008] The degradation of casein mediated by microorganisms has been described mainly in the bacterial genus Lactobacillus, genus known to be frequently found in dairy products. Among the species of this genus that are known for their ability to degrade casein in dairy products, is the bacterium Lactobacillus lactis.

[0009] However, the state of the art does not teach any microbial group such as the one described in the present invention. Specifically, no document described in the state of the art discloses a group of microorganisms associated with this particular use, composed of Lactobacillus sp. strain K03D08, Bacillus sp. strain K03B01 and Kazachstania sp. strain K03K02G, which allows obtaining a dairy derivative with a casein content of less than 0.5% w/v. Additionally, the process described in the present application works optimally at significantly lower temperatures (26° C.-37° C.) compared to the current process (45° C.-60° C.), therefore significant savings are also expected due to this temperature differential.

[0010] Thus, the high cost in terms of the use of enzymatic preparations to obtain dairy products with a low casein content remains as a problem in the state of the art; further, the currently available microorganisms cannot act efficiently to perform this process.

[0015] 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.

[0016] 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.

[0017] 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.

[0018] All the strains described in the present invention, i.e. the Bacillus sp. K03B01 strain, Lactobacillus sp. K03D08 strain and Kazachstania sp. K03K02G strain, have been deposited in the Chilean Collection of Microbial Genetic Resources (CChRGM), at Avenida Vicente Méndez 515,

[0019] Chillán, Región de Ñuble, Chile, in accordance with the Budapest Treaty. Access numbers for each strain are specified below.

[0020]

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

[0021] 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.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] The final number of microorganisms in the composition of the present invention is: the isolated strain of Lactobacillus sp. K03D08 present between 10¹ and 10¹⁵ CFU/ml or CFU/g, the isolated strain of Kazachstania sp. K03K02G is present between 10¹ and 10¹⁵ CFU/ml or CFU/g, and the isolated strain of Bacillus sp. K03B01 is present between 10¹ and 10¹⁵ CFU/ml or CFU/g.

[0026] Furthermore, the present invention discloses a procedure for obtaining each of the microorganisms, and this procedure involves the following steps:

[0027] 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).

[0028] 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.

[0029] 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.

[0030] 4) Centrifuge each culture of microorganisms between 1.000 and 4.000 rpm for 5-30 min at 4-25° C.

[0031] 5) Discard the supernatant and suspend the microbial cells in the same volume of physiological serum (NaCl 0.9% w/v).

[0032] 6) Quantify the optical density of the suspensions obtained in point 5) at a wavelength of 595-600 nm (O.D: 600 nm).

[0033] The present invention further discloses a process for the production of a dairy derivative with a low casein content that comprises the following steps:

[0034] 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.

[0035] b) Preparing a composition comprising the microorganisms Lactobacillus sp. K03D08 and Kazachstania sp. K03K02G in a (1:1) ratio.

[0036] 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.

[0037] d) Centrifugating the solution obtained in point c), between 4.000 and 10.000 rpm and recovering the supernatant.

[0038] e) Filtering the supernatant through a membrane of 1-1000 μm pore diameter.

[0039] f) Filtering the supernatant through a 0.2-0.45 μm pore diameter membrane.

[0040] g) Recovering the filtrate

[0041] 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).

[0042] In a more specific embodiment, the incubation period of milk of bovine origin with Bacillus sp. K03B01 is between 12 and 96 hours.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 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).

[0047]

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

[0048] 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.

[0049]

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

[0050]

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

[0051]

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	+	−	−

[0052]

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.

[0053] 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.

[0054]

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

[0055] 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.

[0056] 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.

[0057]

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	<3 g/100 ml
ND: Not detected

[0058]

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
	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	<3 g/100 ml
	N.D.: Not detected