PL237228B1 - New strain of Lactobacillus hilgardii FLUB bacteria and use of the strain for the production of mannitol - Google Patents

Abstract

The subject of the invention is a new strain of the species Lactobacillus hilgardii FLUB deposited in the International Collection of Cultures of Industrial Microorganisms of the Institute of Biotechnology of the Agricultural and Food Industry in Warsaw under the number KKP 2067p. The subject of the invention is also the use of the Lactobacillus hilgardii FLUB strain for the production of mannitol.

Description

Description of the invention

The subject of the invention is a new strain of Lactobacillus hilgardii FLUB bacteria that effectively produces mannitol.

Mannitol is a naturally occurring polyol that is widely used in the pharmaceutical, food and chemical industries.

Polyols have found their application as sugar substitutes, i.e. sweeteners. Mannitol is poorly absorbed by the human body, and therefore the increase in blood glucose and insulin requirements are much lower than after consuming sucrose. It is only partially absorbed (~ 25%) from the small intestine and is not metabolized. Mannitol is estimated to be half as sweet as sucrose and has the desired cooling effect that is effective in masking bitter tastes. Mannitol can be mixed with other ingredients and sweeteners, which can result in a synergistic effect of better sweetness and flavor. This sugar alcohol is characterized by a pleasant taste, stability, even at high temperatures and a high melting point (165-169 ° C).

It is used as a texturing agent with a sweet taste. Mannitol reduces the tendency to crystallize products, shows a very low hygroscopicity, extends the shelf life of the product. It is most often used as an ingredient in chewing gums. Since mannitol is not hygroscopic, it is used as a filler for sugar-free chewing gum coatings or as a powder coating to prevent gum from sticking to manufacturing equipment or packaging.

Mannitol in the pharmaceutical industry prevents the absorption of moisture from the air, has excellent mechanical properties such as compressibility, does not interact with active ingredients, and its sweet, cool taste masks the unpleasant taste of many drugs. It can also be used as a strong osmotic diuretic and as an antioxidant, it reduces the effects of free radicals and thus protects against the development of colon cancer. Due to all these properties of mannitol, as a food additive it increases their nutritional and health-promoting values.

Mannitol is chemically synthesized by catalytic hydrogenation of a mixture of fructose and glucose at high temperature and pressure. The reaction produces a mixture of mannitol and sorbitol that must be purified, which further increases the price of the end product. The high costs of the substrates used, the physical conditions of the reaction and the need to carry out the difficult process of separating mannitol from sorbitol make the preparation of mannitol by microbiological route extremely attractive on an industrial scale.

Mannitol-producing strains of the genus Lactobacillus are described in international patents. Examples of such patents are: JP6377091A (1991-03-06), EP19910119453 (1991-11-14), US

6,855,526 B2 (2005-02-15), CN 201310111633 (2013-04-01), PCT / CN2014 / 090476 (2014-11-06).

Many microorganisms capable of producing mannitol are known. US Patent Application Publication No. US2009023195 (A1) relates to the biotechnological use of strains from the US ARS Collection (NRRL) among which Lb was selected. intermedius NRRL B-30560 effectively producing mannitol. The primary carbon substrate in both fermentation and biochemical transformations is fructose, but the secondary carbon source may also be glucose, maltose, mannose and galactose.

On the other hand, the following mannitol-producing strains are known from the literature on the subject: Lb. hilgardii NRRL B-1843 and Lb. intermedius NRRL B-3693 (Arena, ME, Landete, JM, Manca de Nadra, MC, Pardo, I., & Ferrer, S. (2008). Factors affecting the production of putrescine from agmatine by Lactobacillus hilgardii XIB isolated from wine. Journal of applied microbiology, 105 (1), 158-165.)

The aim of the invention was to isolate from mead - an environment rich in fructose and other sugars such as glucose, sucrose and maltose, a new strain of bacteria that will effectively produce mannitol outside the cell without genetic modification.

The subject of the invention is a new Lactobacillus hilgardii FLUB strain deposited in the International Collection of Industrial Microbial Cultures of the Institute of Agricultural and Food Biotechnology in Warsaw under the number KKP 2067p.

The invention also relates to the use of the Lactobacillus hilgardii FLUB strain for the production of mannitol.

Preferably, fructose is used as the sole carbon source for the production of mannitol.

Preferably fructose and glucose are used as the carbon source.

Preferably, the addition of each of the fructose sugars and glucose in the culture medium is 4% w / v.

Preferably, glucose-fructose syrup is used as the sole carbon source.

PL 237 228 Β1

Example 1

Strain identification

In order to identify a new isolate, the MALDI-TOF technique was used, the BioTyper program qualified it as Lactobacillus hilgardii with a score of 2.03 which allows for certain identification to the genus and probable assignment to the species. Additionally, the 16S rRNA gene was amplified and sequenced. The alignment of the gene sequence with the NCBI GeneBank database shows 100% agreement of the tested strain with the sequences of the Lb species listed there. hilgardii.

The results of sequencing the 16S rRNA fragment of the Lactobacillus hilgardii FLUB strain determined by the present invention are as follows:

TGCAAGTCGAACGCGTCTTGGTCAATGAAGTTGAGTGCTTGCATTTAACT

GATTTGACATTAAGACGAGTGGCGAACTGGTGAGTAACACGTGGGTAAC

TTGCCCCGAAGCGGGGGATAACATTTGGAAACAGGTGCTAATACCGCAT

AACAACAAAAACCACATGGTTTTTGTTTAAAAGATGGTTTCGGCTATCAC

TTTGGGATGGACCCGCGGCGTATTAGCTAGTTGGTGAGGTAATGGCTCAC

CAAGGCAATGATACGTAGCCGACCTGAGAGGGTAATCGGCCACATTGGG

ACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTC

CACAATGGACGAAAGTCTGATGGAGCAACGCCGCGTGAGTGATGAAGGG

TTTCGGCTCGTAAAACTCTGTTGTTGGAGAAGAACGGGTGTCAGAGTAAC

TGTTGACATCGTGACGGTATCCAACCAGAAAGCCACGGCTAACTACGTGC

CAGCAGCCGCGGTAATCGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGC

GTAAAGCGAGCGCAGGCGGTTTTTTAGGTCTGATGTGAAAGCCTTCGGCT

TAACCGGAGAAGTGCATCGGAAACCGGGAGACTTGAGTGCAGAAGAGG

ACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAA

CACCAGTGGCGAAGGCGGCTGTCTGGTCTGCAACTGACGCTGAGGCTCG

AAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTA

AACGATGAGTGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCT

AACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTC

AAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATT

CGATGCTACGCGAAGAACCTTACCAGGTCTTGACATCTTCTGCTAACCTA

AGAGATTAGGCGTTCCCTTCGGGGACGGAATGACAGGTGGTGCATGGTT

GTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCA

ACCCTTATTGTCAGTTGCCAGCATTTAGTTGGGCACTCTGGCGAGACTGC

CGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCC

TTATGACCTGGGCTACACACGTGCTACAATGGACGGTACAACGAGTCGC

GAAACCGCGAGGTCAAGCTAATCTCTTAAAGCCGTTCTCAGTTCGGATTG

TAGGCTGCAACTCGCCTACATGAAGTTGGAATCGCTAGTAATCGTGGATC

AGCATGCCACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCAC

ACCATGAGAGTTTGTAACACCCAAAGCCGGTGAGGTAACCTTCGGGGGC

CAGCC

PL 237 228 Β1

The described strain of Lactobacillus hilgardii FLUB is characterized by the following morphological features:

Gram-positive, non-spore-producing bacteria are in the form of sticks appearing singly or linked in chains, as shown in Fig. 1 (Scanning electron microscope micrograph of Lb. hilgardii FLUB cells, magnification 15,000) in the attached figure, show growth under anaerobic conditions and relatively aerobically, on MRS (de Man, Ragosa and Sharpe) medium with agar. The colonies are medium-sized, about 1-3 mm in diameter, with a circular, regular shape. The profile of the colony is slightly convex with a smooth, regular margin. The colony is opaque, dull white-cream color. The structure of the colony is greasy-cream, after smearing it is stretched, easily forming a homogeneous suspension in water or medium. The smell emitted by the colonies is perceptible and distinctive, characteristic of fermentation with the addition of ethanol. All the described features make the colonies formed by the strain Lb. hilgardii FLUB are type S.

Strain Lb. hilgardii FLUB isolated from mead differs significantly from Lb. hilgardii isolated from wine, in terms of growth preferences. A strain isolated from mead can grow and multiply in higher sugar concentrations. Fig. 1 shows the growth of both strains on media with 15% and 25% glucose, Lb. hilgardii shows an increase of up to 25% of the sugar concentration in the medium.

Use of carbohydrates by strain Lb. hilgardii FLUB was determined with the Hi-Carbo Kit (HiMedia, Mumbai, India). The strain uses fructose, L-arabinose and D-arabinose, esculin and malonate. The other results are presented in Table 1.

Table 1. Carbohydrate utilization

Carbohydrate Score Carbohydrate Score lactose • inositol - xylose + sorbitol - maltose + man n and this is I. - fructose + adonitol - dextrose - arabitol - galactose + erythritol - raffinose - alpha-methyl-D-g lu cooside - trehalose - rammnosis - melibiosis - cellobiosis - saccharose - melezitosis - L-arabinose + alpha-methyl-D-mannoside - mannose - xiitol - inulin - ONPG - sodium gluconate - esculin + glycerol - D-arabinose - salicin - citrate - dulcitol - malonian +

API-ZYM tests (bioMerieux SA, Marcy l'Etoile, France) were also performed for identification. The results are summarized in Table 2.

PL 237 228 Β1

Table 2. Enzyme profile

No. Enzyme Substrate pH Score 1. control - - 2. alkaline phosphatase 2-naphthylphosphate 8.5 - 3. esterase (C 4) 2-naphthylbutyrate 6.5 + 4. Lipase esterase (C 8) 2-naphthyl caprylate 7.5 + 5. lipase (C 14) 2-naphthyl myristate 7.5 + 6. leucine arylamidase L-leucy lo-2-naphthyllamide 7.5 + 7. valine arylamidase L-wal and il ο-2-kerosene 1 amide 7.5 + 8. cystine arylamidase L-cy sty lo-2-kerosene lam id 7.5 + 9. trypsin N-benzoyl-DL-arginine-2-naphthyllamide 8.5 - 10. a-chymotrypsin N-glutaryl-phenylalanine ο-2-naphthyllamide 7.5 - 11. acid phosphatase 2-naphthyl phosphate 5.4 + 12. Naphthyl-AS-Bl phosphohydrolase Naphthyl AS-BI-phosphate 5.4 + 13. a-galactosidase 6-Br-2-naphthyl-aD-ga! Actopyranoside 5.4 + 14. β-galactosidase 2-naphthyl-3D-galactopyranoside 5.4 + 15. β-glucuronidase Kerosene lo-AS-Bl-pD-glucuronide 5.4 + 16. a-glucosidase 2-naphthyl-αD-glucopyranoside 5.4 + 17. β-glucosidase 6-Br-2-naphthyl-PD-glucopyranoside 5.4 + 18. N-acetates Ιο-β-glucosam and idaza 1-naphthyl-N-acetyl-pD-glucosamine 5.4 - 19. a-mannosidase 6-Br-2-kerosene lo-aD-mannop and ranside 5.4 - twenty. β-fucosidase 2-kerosene lo-aL-iranose fucop d 5.4 -

Isolated from mead, a new strain of Lb. hilgardii FLUB is characterized by osmotolerance, shows resistance to high osmotic pressure caused by the high concentration of sugars in the environment of its occurrence, which is shown in Fig. 2. It may be of significant importance in the industrial production of mannitol using the described Lb strain. hilgardii FLUB. Lb was used as control strains in the studies. hilgardii NRRL B-1843 and Lb. intermedius NRRL B-3693. (Arena et al. 2008, Carvalheiro et al. 2011).

Isolated from mead Lb. hilgardii FLUB produces the most mannitol on a medium with glucose and fructose (8% w / v, in a 1: 1 ratio). After 7 days of cultivation, the presence of mannitol in the culture medium was determined at 4.942% for Lb. hilgardii FLUB, 4.295 for Lb. hilgardii NRRL, and 1.63% for Lb. No. intermedius NRRL. After the fifth day, the amount of mannitol begins to decline in the case of Lb. intermedius NRRL. Contrary to Lb. intermedius NRRL, species Lb. hilgardii did not secondary metabolize the produced mannitol as a carbon source, which, apart from the ability to produce effectively, is a desirable feature. It was also found that Lb. hilgardii FLUB as opposed to Lb. hilgardii NRRL B-1843 has the ability to efficiently biosynthesize mannitol from fructose when fructose is the only carbon source in the medium. In the case of mannitol production using Lb. hilgardii FLUB on a fructose medium, the percentage of this polyol was over 100% higher than in the tested control strains Lb. hilgardii NRRL B-1843 and Lb. intermedius NRRL. When using the 8% fructose medium, generally less mannitol production was observed than when using the medium with a mixture of sugars 4% glucose and 4% fructose. As mannitol is synthesized from fructose by the enzyme mannitol dehydrogenase, cells of all species used in this study use glucose for metabolic purposes and convert fructose into mannitol. The study also checked the production of mannitol from a cheap source of sugar, i.e. glucose-fructose syrup. Here also Lb. hilgardii FLUB produces larger amounts of mannitol in the 7th day - 0.25%, while Lb. hilgardia NRRL, 0.19% on day 7. No. intermedius NRRL did not produce mannitol on this carbon source. This may be due to the fact that glucose-fructose syrup is a mixture of various sugars, not only glucose and fructose, which may result in too high an osmotic concentration for Lb. intermedius NRRL or the presence of other sugars redirects this species to other metabolic pathways. However, the results of the performed studies confirmed the higher efficiency of mannitol production by the described Lb. hilgardii FLUB after 7 days than in other Nos. hilgardii NRRL iLb. intermedius NRRL. The analyzes performed show a greater affinity for fructose in Lb. hilgardii FLUB, which is due to adaptation to

PL 237 228 Β1 the environment in which he lives. The mead from which it was isolated is an unfavorable environment due to alcohol or high sugar concentrations, therefore the Lb. hilgardii FLUB has developed appropriate mechanisms to take over this ecological niche. Its adaptation to an environment rich in sugars, and in particular in fructose, has made it an excellent producer of mannitol.

Example 2

Mannitol production from fructose by strain Lb. hilgardii FLUB

Flasks with MRS medium were inoculated with 2% of the inoculum. Cultures were established on a growth medium with the addition of fructose 8% (w / v). Cultures were carried out for 7 days at 30 ° C with 150 rpm shaking (Minitron Incubator Shaker Infors AG, Switzerland). Culture samples were taken daily and, after biomass centrifugation, the pure post-culture fluid was stored at -20 ° C until further analysis. The percentage of mannitol was determined by HPLC (Gilson Medical Electronics, Villiers-le-Bel, France). The samples were previously purified from proteins using Carrez III reagent. Mannitol concentration was read from the chromatograms using CHROMAX software (POL-LAB). The percentage of mannitol produced by the test strains on each cultivation day is shown in Table 3 with regard to the efficiency of the biosynthetic process and in the graph shown in Fig. 3.

Table 3

days Mannitol in nutrient solution [%] Lactobacillus hilgardii FLUB Lactobacillus hilgardii NRRL Lactobacillus intermedius NRRL AND 0.253 0.230 0.284 II 0.369 0.258 0.325 III 0.556 0.233 0.544 IV 1.673 0.928 0.963 V 1.993 1.112 3.010 VI 2,786 1.329 1,887 VI] 3.698 1.604 1.351

Example 3

Mannitol production from glucose and fructose by strain Lb. hilgardii FLUB

Flasks with MRS medium were inoculated with 2% of the inoculum. Cultures were established on a culture medium with the addition of glucose and fructose 4% w / v of each sugar. Cultures were carried out for 7 days at 30 ° C with 150 rpm shaking (Minitron Incubator Shaker Infors AG, Switzerland). Culture samples were taken daily and, after biomass centrifugation, the pure post-culture fluid was stored at -20 ° C until further analysis. The percentage of mannitol was determined by HPLC (Gilson Medical Electronics, Villiers-le-Bel, France). The samples were previously purified from proteins using Carrez I II reagent. Mannitol concentration was read from the chromatograms using CHROMAX software (POL-LAB). The percentage of mannitol produced by the tested strains on each cultivation day is shown in Table 4 with regard to the efficiency of the biosynthetic process and in the graph shown in Fig. 4.

Table 4

days Mannitol in nutrient solution [% Lactobacillus hilgardii FLUB Lactobacillus hilgardii NRRL Lactobacillus intermedius NRRL AND 0.152 0.181 0.147 II 0.309 0.223 0.284 III 0.506 0.379 0.515 IV 1.933 1.496 1.356 V 2.205 1.962 3.549 VI 3.099 3.120 2.090 VII 4.942 4.295 1.630

PL 237 228 Β1

Example 4

Mannitol production by Lb. hilgardii FLUB with the use of glucose-fructose syrup

Flasks with MRS medium were inoculated with 2% of the inoculum. The cultures were set up in a medium where the only carbon source was 8% (w / v) glucose-fructose syrup. Cultures were carried out for 7 days at 30 ° C with 150 rpm shaking (Minitron Incubator Shaker Infors AG, Switzerland). After 4 and 7 days, culture samples were taken and, after biomass centrifugation, the pure post-culture fluid was stored at -20 ° C until further analyzes. The percentage of mannitol was determined by HPLC (Gilson Medical Electronics, Villiers-le-Bel, France). The samples were previously purified from proteins using Carrez III's reagent. Mannitol concentration was read from the chromatograms using CHROMAX software (POL-LAB). The percentage of mannitol produced by the test strains on each cultivation day is shown in Table 5 with regard to the efficiency of the biosynthetic process and in the graph shown in Fig. 5.

Table 5

Days Mannitol in bNutrient [%] Lactobacillus hilgardii FLUB Laciobacillus hilgardii NRRL ] 0.082 0.055 AT 0.124 0.098 III 0.153 0.149 JV 0.186 0.188 V 0.192 0.189 V! Oh, 22i 0.191 VII 0.249 0.194

Patent claims

Claims (6)

1. A new strain of Lactobacillus hilgardii FLUB bacteria deposited at the International Collection of Industrial Microbial Cultures of the Institute of Agricultural and Food Biotechnology in Warsaw under the number KKP 2067p. 2. Use of the strain as defined in claim 1 for the production of mannitol, 3. Use according to claim 1 The process of claim 2, wherein the sole carbon source is fructose. 4. Use according to claim 1 The process of claim 2, wherein the carbon source is fructose and glucose. 5. Use according to claim 1 4. The process according to claim 4, characterized in that the addition of each of fructose sugars and glucose in the culture medium is 4% w / v each. 6. Use according to claim 1 2. The process of claim 2, wherein the sole carbon source is glucose-fructose syrup.