Patent Publication Number: US-2023138227-A1

Title: 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

Description:
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. 
     BACKGROUND AND PRIOR ART 
     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). 
     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. 
     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. 
     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&#39;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. 
     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. 
     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.    
     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. 
     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. 
    
    
     
       BRIEF DESCRIPTION OF FIGURES 
         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. 
         FIG.  2   . Gram stain of selected microbial strains. A:  Bacillus  sp. strain K03B01. B:  Lactobacillus  sp. strain K03D08. C:  Kazachstania  sp. strain K03K02G. 
         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. 
         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 
     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. 
     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. 
     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. 
     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. 
     
       
         
           
               
               
               
             
               
                   
               
               
                 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 
               
               
                   
               
            
           
         
       
     
     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. 
     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. 
     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. 
     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. 
     The final number of microorganisms in the composition of the present invention is: the isolated strain of  Lactobacillus  sp. K03D08 present between 10 1  and 10 15  CFU/ml or CFU/g, the isolated strain of  Kazachstania  sp. K03K02G is present between 10 1  and 10 15  CFU/ml or CFU/g, and the isolated strain of  Bacillus  sp. K03B01 is present between 10 1  and 10 15  CFU/ml or CFU/g. 
     Furthermore, the present invention discloses a procedure for obtaining each of the microorganisms, and this procedure involves the following steps: 
     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). 
     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. 
     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. 
     4) Centrifuge each culture of microorganisms between 1.000 and 4.000 rpm for 5-30 min at 4-25° C. 
     5) Discard the supernatant and suspend the microbial cells in the same volume of physiological serum (NaCl 0.9% w/v). 
     6) Quantify the optical density of the suspensions obtained in point  5 ) at a wavelength of 595-600 nm (O.D: 600 nm). 
     The present invention further discloses a process for the production of a dairy derivative with a low casein content that comprises the following steps: 
     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. 
     b) Preparing a composition comprising the microorganisms  Lactobacillus  sp. K03D08 and  Kazachstania  sp. K03K02G in a (1:1) ratio. 
     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. 
     d) Centrifugating the solution obtained in point c), between 4.000 and 10.000 rpm and recovering the supernatant. 
     e) Filtering the supernatant through a membrane of 1-1000 μm pore diameter. 
     f) Filtering the supernatant through a 0.2-0.45 μm pore diameter membrane. 
     g) Recovering the filtrate 
     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). 
     In a more specific embodiment, the incubation period of milk of bovine origin with  Bacillus  sp. K03B01 is between 12 and 96 hours. 
     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. 
     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 
     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. 
     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 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 
               
               
                   
               
            
           
         
       
     
     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 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 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 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 5 
               
             
            
               
                   
               
               
                 Casein hydrolysis in selected strains 
               
            
           
           
               
               
               
            
               
                   
                 Hydrolysis 
                   
               
               
                   
                 halo in skim 
                 Casein 
               
               
                 Strain 
                 milk agar (mm) 
                 Quantification* 
               
               
                   
               
            
           
           
               
               
               
            
               
                   Bacillus  sp. K03B01 
                 60 
                 &lt;0.00025 g/100 ml   
               
               
                   Lactobacillus  sp. K03D08 
                 15 
                 &gt;2.5 g/100 ml 
               
               
                   Kazachstania  sp. K03K02G 
                 0 
                 &gt;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 
     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 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: &lt;0.00025 g/100 ml 
                 Casein: &lt;0.00025 g/100 ml 
                 Casein: &lt;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: &lt;3 g/100 ml 
               
               
                   
               
               
                 N.D.: Not detected 
               
            
           
         
       
     
     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 
     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 7 
               
             
            
               
                   
               
               
                 Characterization of the dairy derivative generated through the different production processes 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   Bacillus  sp. strain 
                   Bacillus  sp. strain 
               
               
                   
                 
                   Bacillus 
                 
                 K03B01: Lactobacillus sp. 
                 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 
                 &lt;0.00025 g/100 ml 
                 &lt;0.00025 g/100 ml 
                 &lt;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 
                     &lt;3 g/100 ml 
               
               
                   
               
               
                 ND: Not detected 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 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 
                 &gt;2.5 g/100 ml 
                 &lt; 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 
                     &lt;3 g/100 ml 
               
               
                   
                   
               
               
                   
                 N.D.: Not detected