FIELD OF THE INVENTION

This invention concerns new strains of Lactobacillus delbrueckii ssp. bulgaricus (Selur 6 and Selur 19) and Streptococcus thermophilus (Selur 12), and the use of these new strains in combination with the probiotic strain Lactobacillus gasseri K7 to produce probiotic food supplements intended for use by humans or animals. This invention also refers to prophylactic and therapeutic preparations containing as active component various combinations of the above-mentioned strains, intended to preserve and improve the general health status of their host, as well as to prevent and/ or treat gastro-intestinal tract diseases. Furthermore, new procedures and technologies for the production of biomass, probiotic starter cultures, food supplements and pharmaceutical preparations based on the fermentation of milk or whey from the above-mentioned strains have also been invented and presented.

STATE OF THE ART

Lactic acid bacteria (LABs) are a diverse group of gram-positive, non- sporulent bacteria, anaerobs or facultative anaerobs requiring specific nutrition environment, and whose ultimate metabolite product is lactic acid. Currently, LABs are microorganisms of industrial importance for their fermentation activity, as well as for their health and nutrition benefits. LABs, particularly lactic bacteria, which occupy important niches in the gastro-intestinal tract (GIT) are considered as offering numerous probiotic benefits to general health status and proper physical condition. These benefits include positive effect on natural microflora, competitive exclusion of pathogens and stimulation/modulation of mucous immunity. In this regard, it deserves particular mentioning that the health benefits due to probiotic bacteria are strain-specific - depending on individual strain features (Shah, 2007).

Probably, the most important and sufficiently well studied biogenic substances contained in milk products are proteins from whey and bioactive .peptides..Proteins from, whey are involved. in numerous nutritious, and . physiological ... results, including physiological changes, restoration after physical exercise and prevention of muscular atrophy, feeding-up and weight control, infection and health control with age progress (Smithers, 2008). Milk proteins are a rich source of bioactive peptides. Bioactive peptides originating from milk proteins are not active in parent protein sequence and may be released from LABs by enzyme proteolysis during milk/ whey digestion or fermentation. Once released, bioactive peptides may act in the body as regulatory compounds of various activity, such as: means for decrease or control of high blood pressure, anti-oxidants, means for prevention of thrombi formation, anti-microbial means and means for immune system regulation (Meisel, 1998, Korhonen and Pihlanto, 2006).

Yoghurt is the most popular of all types of milk produced as a result of fermentation. The belief in its beneficial effect on human health has been shared by many civilizations over a long period of time. Ayurveda, one of the most ancient health sciences, which appeared some 2,500 years B.C., recommended consumption of yoghurt to preserve proper general health status (Chopra &. Doiphode, 2002). Notwithstanding the fact that yoghurt, and therefore, yoghurt bacteria Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus have been historically related with human health, they are not mentioned often as probiotic bacteria. The reason for this circumstance lied most probably in the inability to observe these bacteria in the natural microflora of human gastro-intesrinal tract (GIT) and therefore, in the past, most scholars believed that these species do not survive in the conditions of the GIT and the tract (Guarner et al., 2005). The development of genetic methods furthered scholars to trace some specific strains during their transition through the GIT. Recently, lactic acid bacteria have been observed in the excrements of humans consuming yoghurt (Mater et al., 2005, Elli et al., 2006). On the opposite, Garcia- Albiach et all. (2008) suggest that the modifications in human faecal microorganisms are usually due to the properties of yoghurt itself, which does not require bacteria viability. In this respect, patent applications WO 96/20607, WO 2005/056028 and WO 2008/002484 reveal specific strains of a starter culture for yoghurt (Str. I ^' hennophilus and -Lb: bulgaricus) with probiotic. properties, their combinations and-, preparations for treatment of various GIT disorders.

Until 1980, most of the Lactobacillus strains isolated from intestines were classified as Lb. acidophilus based on their morphological and phenotype characteristics. With the development of modern taxonomy based on molecular techniques, six different species in the group of the previously determined Lb. acidophilus were identified which were then divided into A (Lb. acidophilus, Lb. amylovorus, Lb. crispatus, Lb. gallinarum) and B (Lb. gasseri, Lb. johnsonii) DNA homologous groups. It was established that the hyper-variable 16S-23S intergene spacer regions, as well as the 16S rPHK gene sequences are specific enough to differentiate between similar Lactobacillus strains, and through the use of the type- specific PCR primers or through the DNA-sequence (Tannock et al., 1999, Kullen et al., 2000).

The Lb. gasseri K7 strain was isolated from a healthy 7-day-old breast-fed baby at the Chair of Dairy Science, Biotechnical Faculty, University of Ljubljana, in 1996. It was" deposited in the ZIM Culture Collection of Industrial Microorganisms, registered as WDCM810 (WFCC-MIRCEN World Data Centre for Microorganisms (WDCM)). In September 2009, it was deposited based on the Budapest Treaty in the Czech Collection of Microorganisms (CCM) under Registration Number CCM 7710. Based on the physiological and biochemical properties, including identification of the fermentation schemes by API 50 CHL (BioMerieux, France) it was determined as member of the Lb. acidophilus group. The lack of S-layer protein, PCR with strain- specific primers and determination of the primary sequence of V2-V3 regions of 16S rflHK has shown that strain K7 belongs to the type of Lb. gasseri (Bogovi£ MatijaSic M Rogelj, 2000, Canzek Majhenie et al., 2003).

It has been shown that Lb. gasseri K7 complies with the basic criteria for probiotic strains, since it is resistant to low pH and bile liquid, produces antimicrobial substances, including bacteriocenes with wide anti-microbial spectrum and attaching to the Caco-2 cells [Bogovii MatijaSic M Rogelj, 2000, Bogovii Matijas c et al., 2003]. The specific about Lb. gasseri K7 is the biosynthesis and production of at least tw.o,bacteriocenes. called gassericene K7..A and . gassericene. K7. B, whose nucleotide sequences were deposited in the GenBank under Registration Numbers EF392861 and AY307382. Both bacteriocenes belong to the group of the two-peptide bacteriocenes (Zorii Peternel, 2007).

In addition, the strain is capable to survive, passing through the gastrointestinal tract, and to form colonies in the intestinal ligament of little pigs born in the standard way or by Caesarean section, at least temporarily. In little pigs born by Caesarean section and infected artificially with the enterotoxigenous Escherichia coli, Lb. gasseri K7 decreased up to a certain range the infection's survival and apart from the anti-microbial activity due to the production of organic acids and stimulation of the immune response, the competitive release of E. coli from the intestinal ligament served as a possible mechanism for such activity. The ability of Lb. gasseri K7 to protect enterocyte colonization by the non-enterotoxigenous mutant E. coli strain 08:K88 ⁺ was confirmed by cellular models Caco-2 (Bogovii MatijaSid et al., 2004, Bogovii Matijagic et al., 2006, Rogelj and Bogovii MatijaSic, 2006). Some earlier patent applications describe the isolation of a.. set of various bacterial strairis, including probiotic Lb. Gasseri strains from babies' faeces (JP 04320642, JP 05227946, US patent 6596530) and human milk (WO 2004/003235, WO 2008/016214)

Furthermore, USA Patent Application US2008233104 reveals compositions and methods for application of probiotic organisms in therapeutic compositions. In particular, the use of one or more strains of lactic acid bacteria to control pathogenes is revealed. Bacterial strains possess resistivity or strongly reduced sensitivity to antimicrobial means, such as antibiotics and anti-viral means. The revealed compositions are used for treatment or prevention of bacteria-mediated infections of the gastrointestinal tract.

International Patent Application WO8905849 reveals lactic acid bacteria isolated from the gastro-intestinal tract of pigs for their ability to survive in gastrointestinal tract medium, i.e. for their bile or acid tolerability. These bacteria are included in a fermented milk product intended for consumption by humans or animals to treat or prevent diseases of the gastro-intestinal tract. ·... - NZ523010. reveals an isolated. strain of lactic acid bacteria possessing probiotic activity. The strains have been chosen among Lactobacillus, Bifidobacterium or Enter ococcus, and more precisely, Lactobacillus reuteri NCC2581 (CNCMI-2448), Lactobacillus reuteri NCC2592 (CNCM 1-2450), Lactobacillus rhamnosus NCC2583 (CNCMl-2449), Lactobacillus reuteri NCC2603 (CNC I-2451), Lactobacillus reuteri NCC2613 (CNCMI-2452), Lactobacillus acidophilus NCC2628 (CNCM 1-2453).

DETAILED DESCRIPTION OF THE INVENTION

This invention concerns strains of Lactobacillus delbrueckii ssp. bulgaricus (Lb. bulgaricus), and Streptococcus thermophilus (St. Thermophilus), isolated from traditional yoghurt produced in Bulgarian mountains - Stara Planina and Rhodopes, which demonstrate several unique characteristics. These strains are:

• Lb. bulgaricus Selur 6, which was deposited in September 2009 according to the Budapest Treaty in the Czech Collection of Microorganisms (CCM) under Registration Number CCM 7712,

IK L&. bulgaricus Selur 19, which Ϊ was deposited in September 2009 accdrdmg to the Budapest Treaty in the Czech Collection of Microorganisms (CCM) under Registration Number CCM § j |

§§fjSf. Thermophilus Selur 12, which was deposited in September 2009 according to the Budapest Treaty in the Czech Collection of Microorganisms (CCM) under Registration Number CCM 7711.

Another aspect of the invention > concerns compositions and products, containing at least one of the above-mentioned strains, with the above-mentioned probiotic Lb. gasseri K7 strain, isolated from child's faeces, with Registration Number CCM 7710.

Another aspect of the invention reveals procedures for the production of biomass and probiotic starter cultures, food supplements for humans and animals and pharmaceutical preparations based on the fermentation of milk or hey from the above-mentioned strains. .. ^, - ^, -... ^, , -Finally- _; - -the- -last- -aspeet- )f -.t¾e--irivention-.con6ems...the..use, .of --the above- . mentioned strains or any culture, composition or product, which contains them, during the production of functional food products and prophylactic preparations, such as food supplements for humans and animals and therapeutic preparations for preservation of the host's general health status, as well as for prevention and/ or treatment of gastro-intestinal tract disorders, uro-genital infections or vaginal infections.

Definitions

According to the invention, the term„probiotic" should be understood as defined by the FAO/WHO (2002), and in particular, these are live organisms which, when ingested in appropriate quantities, bring certain physiological benefit, and are usually strain-specific.

According to the invention, the term„biogene substances" means nutrition components originating from microbial activity, which are beneficial to health status, not involving intestinal microflora (Mitsuoka, 2000). According to this definition, such substances from fermented milk are for instance, lactic acid, butyric acid, bioactive peptides, β-galactosidase and exopolysaccharides produced from LAB during fermentation.

According to the invention and, as used here, the term "nutrition and/ or pharmaceutically acceptable substances/ bearer" means one or more solid or liquid substances or fillers, diluents or capsulating substances, which is(are) suitable for , intake by humans or animals and which is(are) compatible with the active probiotic strain.

According to the invention, the term "compatible" refers to components, which are not inhibiting to probiotic bacteria and allow chestnut colouration and strain activity according to this invention and the Lb. gassen strain 7 in the human or animal body.

BRIEF DESCRIPTION OF THE DRAWING Figure 1 shows PCR with Lb. delbrueckii type-specific Del I and II primers. Key : M - lOObp (Ferments);,!..Selur 6;_2, LZ; 3. L8;A. L12; 5. Selur.l9;,6. LMG 6412 ^T , Lb. , delbr.bulg.; 7. LMG 6901 ^T Lb. delbr.delbr.; 8-12 Lb.delbr.bulg. isolated from natural yoghurts; 13. Negative control;

Figure 2 shows PCR with Str. thermophilus type-specific Thl and Th2 primers.

Key: M - lOObp (Ferments); 1 - ST4; 2 - ST7; 3 - Selur 12

Figure 3 shows PCR with Lb. delbrueckii subsp. bulgaricus subtype-specific LB1 M LLB1 primers. Key: M - lkb (Ferments); 1 - Selur 6; 2 - L7; 3 - L8; 4 - L12; 5 - Selur 19; 6 - LMG 6412 ^T Lb. delbr.bulg.; 7 - LMG 6901 ^T Lb. delbr.delbr.; 8-12 - Lb.delbr.bulg.

isolated from natural yoghurts; 13. Negative control.

Figure 4 shows DNA fragments obtained after RAPD-PCR with primer 1254.

Key: M - lkb (Ferments); 1 - Selur 6; 2 - L7; 3 - L8; 4 - L12; 5 - Selur 19; 6 - LMG 6412 ^T Lb. delbr.bulg.; 7 - LMG 6901 ^T Lb. delbr.delbr.; 8-12 - Lb.delbr.bulg. isolated from natural yoghurts; 13. Negative control.;

Figure 5 shows DNA fragments obtained after RAPD-PCR with primer KGT- 70GC. Key: 1+12 - Marker 1 kb (Ferments); 2 - Lb. gassen K7; 3 - Str. thermophilus Selur 12; 4 - Lb. bulgaricus Selur 6; 5 - Lb. bulgaricus Selur 19; 6-10 - Lb.delbr.bulg. isolated from natural yoghurts; 11 - Negative control (RAPD - reaction reference mixture).

Figure 6 presents antimicrobial activity of Lb. bularicus Selur 6 and Selur 19 strains against Staphylococcus aureus ATCC 29213. Lb. gasseri K7 was adopted for comparison.;

Figure 7 presents the adhesive, ability of various strains to CaCo-2 cells; (1 = Lb. bulgaricus Selur 19, 2 = Str. thermophilus Selur 12, 3 = Lb. bulgaricus Selur 6, 4 = Lb. rhamnosus GG, 5 = Lb. johnsonii Ljl, 6 = Lb. reuteri ING 1, 7 = Lb. Shirota SHI, 8 = Lb. gasseri K7).

Figure 8 presents the change of pH values during the fermentation of milk infected Str. thermophilus Selur 12 and various lactobacilli strains (Selur 6, Selur 19 n K7) npw 42°C M 37°C * DETAILED DESCRIPTION OF THE INVENTION

New Lactobacillus delbrueckii ssp. bulgaricus strains Selur 6 and Selur 19 and Streptococcus thermophilus strain Selur 12

Specimen of the new Lactobacillus delbrueckii ssp. bulgaricus strains Selur 6 and Selur 19 and Streptococcus thermophilus strain Selur 12 have been deposited in the CCM (Czech Collection of Microorganisms) under Registration Numbers CCM 7712, CCM 7713, and CCM 7711 with deposition date September 2009. The deposition was made under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of the Patent Procedure.

Lactobacillus strain Selur 6 and Streptococcus strain Selur 12 were isolated from traditional yoghurt obtained from raw milk from the Stara Planina Mountain (Bulgaria) and Lactobacillus strain Selur 19 was isolated from traditional yoghurt obtained from raw milk from the Rhodopes Mountain (Bulgaria).

In the same way, -^specimen of Lactobacillus gasseri strain K7 was. deposited in the CCM (Czech Collection of Microorganisms) under Registration Number CCM 7710 with deposition date September 20092009. This deposition was re-made under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of the Patent Procedure. Lb. gasseri K7 is a well described probiotic strain (Bogovii MatijaSic and Rogelj, 2000; Bogovii MatijaSic et al., 2003, 2004, 2006; Rogelj and Bogovii MatijaSic, 2006, Zorii Peternel, 2007) with Registration Number IM105 in ' 'the ZIM Culture Collection of Industrial Microorganisms (Slovenia), registered as WDCM810 (WFCC-MIRCEN World Data Centre for Microorganisms (WDCM). It was isolated in August 1996 in Ljubljana, Slovenia, from the faeces of 7-day-old breast-fed babies. K7 is producer of two bacteriocenes: gassericene K7 A and gassericene K7 B. Based on the nucleotide sequences of chromosome bacteriocene operons, both bacteriocenes were classified in the group of two-peptide non-antibiotics (lib). The nucleotide sequences of gassericene K7 A (1143 bp) and gassericene K7 B (3276 bp) were deposited in GenBank under Registration Numbers EF392861 (gassericene K7A) and AY307382 {gassericene - 7B). It contains 1 plasmide . and. forms, shorter, rods located in the long chains. The optimal growth temperature is 37°C in microaerophyllic conditions.

As described in Example 1 below, the new Lactobacillus and Streptococcus strains were first characterised by genotyping. PCR with Lb. delbrueckii strain-specific primers Del I (5'-ACG GAT GGA TGG AGA GCA G-3') and Del II (5 -GCA AGT TTG TTC TTT CGA ACT C-3 ¹ ) and Str. thermophilus strain-specific primers Th I (5'-ACG GAA TGT ACT TGA GTT TC-3') and Th II (5'-TTT GGC CTT TCG ACC TAA C-3') (Tilsala-Timisjarvi M Alatossava, 1997) was carried out on DNA isolated from frequent cultures using Wizard ^® set for purification of genomic DNA (Promega) or using Maxwell™ 16 system (Promega). The sub-type identification of Lb. delbrueckii was carried out with primers LB1 (5'-AAA AAT GAA GTT GTT TAA AGT AGG TA- 3') and LLB1 (5'-AAG TCT GTC CTC TGG CTG G-3') (Torriani et al., 1999). Strains Selur 6 and Selur 19 were identified as Lactobacillus delbrueckii ssp. bulgancus and strain Selur 12 - as Streptococcus thermophilus.

To confirm additionally type identification, sequencing of PCR amplified variable regions VI and ^ 3 of 16S rPHK was carried out. Lactobacillus strains Selur 6 and Selur 19 were identical (99%) with the reference sequence of Lactobacillus delbrueckii ssp. bulgaricus ATCC BAA-365 or ATCC 11842 ((Sequence of PCR extended variable regions VI and V3 of 16S rPHK of Lb. bulgaricus strain Selur 6 (The sequencing was made by Microsynth Lab in Balgach - Switzerland) and Sequence of PCR extended variable regions VI and V3 of 16S rPHK of Lb. bulgancus strain Selur 19 (The sequencing was made by Microsynth Lab in Balgach - Switzerland)),

Al, Premium RUN

986446 49880

GATTTGTTGGACGCTAGCGGCGGATGGGTGAGTAACACGTGGGCAATCTGCC

CTAAAGACTGGGATACCACTTGGAAACAGGTGCTAATACCGGATAACAACA

TGAATCGCATGATTCAAGTTTGAAAGGCGGCGYAAGCTGTCACTTTAGGATG

AGCCCGCGGCGCATTAGCTAGTTGGTGGGGTAAAGGCCTACCAAGGCAATG

ATGCGTAGCCGAGTTGAGAGACTGATCGGCCACATTGGGACTGAGACACGG CCCAAACTCCTACGGGAGGCAGGAG _. TAGGGAAXCTTCCACAAXGGACGCAA. GTCTGAXGGAGCAACGCCGCGXGAGXGAAGAAGGXTXXCGGAXCGXAAAGCX CXGTXGXXGGXGAAGAAGGAXAGAGGCAGXAACXGGXCXXXAXXXGACGGTA ATCAACCAGAAAGXCACGGCXAACXACGXGCCAGCAGCCGCGGXAAXACGX AGGXGGCAAGCGXXGXCCGGAXXXAXXGGGCGXAAAGCGAGCGCAGGCGGA AXGAXAAGXCXGAXGXGAAAGCCCACGGCXCAACCGXGGAACXGCAXCGGAA ACXGXCAXXCXXGAGXGCAGAAGAGGAGAGXGGAAXXCCAXGXGXAGCGGXG AAAX

Xactobacillus 116 hits 20 orgs [root; cellular

organisms; Bacteria; Firmicutes; Bacilli; Xactobacillales; Xactobacillaceae]

. Xactobacillus delbrueckii 94 hits 7 orgs

. . . Xactobacillus delbrueckii subsp. bulgaricus AXCC BAA -365 . 9 hits 1 orgs

. . . Xactobacillus- delbrueckii- subsp. bulgaricus AXCC 11842 :.r 10 hits 1 Orgs

. . Xactobacillus delbrueckii subsp. indicus 1 hits 1 orgs

. . Xactobacillus delbrueckii subsp. delbrueckii 4 hits 1 orgs

. . Xactobacillus delbrueckii subsp. lactis 13 hits 1 orgs

. Xactobacillus sp. JCM 8653 1 hits 1 orgs

. Xactobacillus sp. CYl 1 hits 1 orgs

. uncultured Xactobacillus sp 9 hits 1 orgs [environmental samples]

. Xactobacillus sp. RA2120 1 hits 1 orgs

. Xactobacillus sp. RA2062 1 hits 1 orgs

. Xactobacillus sp. RA2066 1 hits 1 orgs

. Xactobacillus sp. DJF_CR11 1 hits 1 orgs

. Xactobacillus sp. 19-2 1 hits 1 orgs rLactobacillus-sp.' JCM 1552 .r.. hits 1 orgs

. Lactobacillus sp. DumLacl 1 hits 1 orgs

. Lactobacillus sp. MF-07 1 hits 1 orgs

. Lactobacillus sp. DI70 1 hits 1 orgs

. Lactobacillus helveticus 2 hits 1 orgs or

Al, Premium RUN

986445 49879

GATTTGTTGGACGCTAGCGGCGGATGGGTGAGTAACACGTGGGCAATCTGCC

CTAAAGACTGGGATACCACTTGGAAACAGGTGCTAATACCGGATAACAACA

TGAATCGCATGATTCAAGTTTGAAAGGCGGCGTAAGCTGTCACTTTAGGATG

AGCCCGCGGCGCATTAGCTAGTTGGTGGGGTAAAGGCCTACCAAGGCAATG

ATGCGTAGCCGAGTTGAGAGACTGATCGGCCACATTGGGACTGAGACACGG

GTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAGCT

CTGTTGTTGGTGAAGAAGGATAGAGGCAGTAACTGGTCTTTATTTGACGGTA

ATCAACCAGAAAGTCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGT

AGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGA

ATGATAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAACTGCATCGGAA

ACTGTCATTCTTGAGTGCAGAAGAGGAGAGTGGAATTCCATGTGTAGCGGTG

A

Lactobacillus 116 hits 20 orgs [root; cellular organisms; Bacteria; Firmicutes; Bacilli; Lactobacillales; Lactobacillaceae]

. Lactobacillus delbrueckii 94 hits 7 orgs . . . Lactobacillus delbrueckii-subsp.- bulgaricus ATCC BAA-365... . , 9 hits _νΛ 1 orgs

. . . Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 ... 10 hits 1 orgs

. . Lactobacillus delbrueckii subsp. indicus 1 hits 1 orgs

. . Lactobacillus delbrueckii subsp. delbrueckii 4 hits 1 orgs

. . Lactobacillus delbrueckii subsp. lactis 13 hits 1 orgs

. Lactobacillus sp. JCM 8653 1 hits 1 orgs

. Lactobacillus sp. CY1 1 hits 1 orgs

. uncultured Lactobacillus sp 9 hits 1 orgs [environmental samples]

. Lactobacillus sp. RA2120 1 hits 1 orgs

. Lactobacillus sp. RA2062 1 hits 1 orgs

. Lactobacillus sp. DJF_CR11 1 hits 1 orgs

. Lactobacillus sp. RA2066 1 hits 1 orgs

. Lactobacillus sp. 19-2 1 hits 1 orgs

. Lactobacillus sp. JCM 1552 1 hits 1 orgs

. Lactobacillus sp. DumLacl 1 hits 1 orgs

. Lactobacillus sp. MF-07 1 hits 1 orgs

. Lactobacillus sp. DI70 1 hits 1 orgs

. Lactobacillus helveticus 2 hits

1 orgs,

Selur 12 strain was identical (100%) with the reference sequence of Streptococcus thermophilus LMG 18311 ( (Sequence of PCR extended variable regions VI and V3 of 16S rPHK of Str. thermophilus strain Selur 12 (The sequencing was made by

Microsynth Lab in Balgach - Switzerland)),

Al, Premium RUN

98644749881 CJAG ll ^' GCGAACGGGTGAG TAACGCG I AGGTAACC GCCTTGTAGCGGGGGAT " ·- -

AACTATTGGAAACGATAGCTAATACCGCATAACAATGAATGACTCATGTCAT

TTATTTGAAAGGGGCAATTGCTCCACTACAAGATGGACCTGCGTTGTATTAGC

TAGTAGGTGAGGTAACGGCTCACCTAGGCGACGATACATAGCCGACCTGAG

AGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAG

GCAGCAGTAGGGAATCTTCGGCAATGGGGGCAACCCTGACCGAGCAACGCC

GCGTGAGTGAAGAAGGTTTTCGGATCGTAAAGCTCTGTTGTAAGTCAAGAAC

GAGTGTGAGAGTGGAAAGTTCACACTGTGACGGTAGCTTACCAGAAAGGGA

CGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTCCCGAGCGTTGTC

CGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTGATAAGTCTGAAGTTA

AAGGCTGTGGCTCAACCATAGTTCGCTTTGGAAACTGTCAAACTTGAGTGCA

GAAGGGGAGAGTGGAATTCCATGTGTAGCGGTGAAATGCG

Streptococcus 100 hits 4 orgs [root; cellular organisms; Bacteria;

Firmicutes; Bacilli; Lactobacillales; Streptococcaceae]

. uncultured Streptococcus sp. . 46 hits 1 orgs [environmental samples]

. Streptococcus salivarius 10 hits 1 orgs

. Streptococcus sp. C165 1 hits 1 orgs

. Streptococcus thermophdus ... > 43 hits i;brgs

Strain K7 was identical (100%) with the reference sequence of Lactobacillus gasseri

ATCC 33323 ((Sequence of PCR extended variable regions VI and V3 of 16S rPHK of Lb. gasseri strain Selur K7 (The sequencing was made by Microsynth Lab in Balgach - Switzerland)).

Al, Premium RUN

-

AATTTGGTGCTTGCACCAAATGAAACTAGATACAAGCGAGCGGCGGACGGG TGAGTAACACGTGGGTAACCTGCCCAAGAGACTGGGATAACACCTGGAAAC AGATGCTAATACCGGATAACAACACTAGACGCATGTCTAGAGTTTAAAAGAT GG TCTGGTATG'AGTGTTGeATGGAGCTGGGGTGGATTAGGTAGT GGTAAGG

TAACGGCTTACCAAGGCAATGATGCATAGCCGAGTTGAGAGACTGATCGGCC

ACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGA

ATCTTCCACAATGGACGCAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGA

AGGGTTTCGGCTCGTAAAGCTCTGTTGGTAGTGAAGAAAGATAGAGGTAGTA

ACTGGCCTTTATTTGACGGTAATTACTTAGAAAGTCACGGCTAACTACGTGCC

AGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGT

AAAGCGAGTGCAGGCGGTTCAATAAGTCTGATGTGAAAGCCTTCGGCTCAAC

CGGAGAATTGCATCAGAAACTGTTGAACTTGAGTGCAGAAGAGGAGAGTGG

AACTCCATGTGTAGCGGTGAAATG

Lactobacillus Ill hits 19 orgs [root; cellular organisms; Bacteria;

Firmicutes; Bacilli; Lactobacillales; Lactobacillaceae]

. Lactobacillusigasseri .. ,: _v ¾hifs_ 2 rgs

. . Lactoba illus gasseri ATCC 33323 . 7 hits 1 orgs

. Lactobacillus sp. IDS Ac 1 hits 1 orgs

. uncultured Lactobacillus sp 11 hits 1 orgs [environmental samples]

. Lactobacillus sp. BLB3 1 hits 1 orgs

. Lactobacillus sp. KC36a 1 hits 1 orgs

. Lactobacillus sp. BLBlb 1 hits 1 orgs

. Lactobacillus johnsonii 34 hits 2 orgs

. . Lactobacillus johnsonii NCC 533 .. 6 hits 1 orgs

. Lactobacillus sp. BCRC 17755 1 hits 1 orgs

. Lactobacillus sp. 20326H4L1 1 hits 1 orgs

. Lactobacillus sp. DJF_SLA41 1 hits 1 orgs

. Lactobacillus sp. AD102 1 hits 1 orgs

. Lactobacillus sp. L-YJ 1 hits 1 orgs - ^■ - ^"* · ^■· Lactobacillus 'spv- €45a*:r.^-. 2 hits- 1 orgs

. Lactobacillus acidophilus 1 hits 1 orgs

. Lactobacillus sp. JCM 2010 1 hits 1 orgs

. Lactobacillus sp. ID9203 1 hits 1 orgs

Lactobacillus sp. MF213 1 hits 1 orgs

Furthermore, as revealed in Example 1, the RAPD models of the new strains have been determined. It is important to differentiate the special strain from the other, and in this connection RAPD-PCR may be a useful method. To differentiate the new strains from the various strains used in yoghurt's industrial production, several oligonucleotide primers were used. Most suitable for strain differentiation appeared to be RAPD primers 1254 (Fig. 4) or KGT-70GC (Fig. 5). As with both primers, using the described protocol, differentiation between the new Lb. bulgaricus strains (Selur 6 and Selur 19) can be made as well.

In another aspect, this invention describes the characteristics displayed by . 'the new strains on their, own or in combination with Lb. gasseri strain K7, which is beneficial to human health, and in particular, to the prophylaxis or treatment of digestive, infectious or other immune-related diseases, such as allergies or inflammatory diseases, diarrhoea with travellers or diarrhoea due to antibiotic treatment.

As shown in Example 2, various tests were carried out according to Final Technical Report for FSA Project ref. G01022" (FSA, 2005) for evaluation of the new strains' ability to survive in simulated gastro-intestinal tract conditions. According to the results, at pH 1, Selur 6 and Selur 12 were the only strains that survived on 20- minute processing in SCJ to an extent comparable with strain Lb. gasseri K7. At pH 2, where approximately half of the live cells of all tested strains and 100% of the Lb. gasseri K7 cells survived (Table 1). The strains were rather more sensitive to simulated upper intestinal juice composed of bile salts (1 g/1) in PBS buffer (pH 8) and pancreatic enzyme (1 g/L). A decrease in the number of cells of the order of three or four logarithmic units immediately upon exposure to UIJ (at T 0) was observed Jor all tested, strains in pure cultures. Str. thermophilus Selur..12 was not affected immediately, but incubation for 2 hours was fatal for this strain (Table 2).

Strains survived better in preparation cultivated on milk environment (Table 3).

According to the in vitro tests, it was barely probable for Str. thermophilus Selur 12 to survive in GIT conditions. The important characteristic of this strain is its stimulating effect on growth and fermentation ability of the Lb. bulgaricus strains Selur 6 and Selur 19 (as shown in Example 9).

In Example 3, the antibiotic sensitivity of the new strains, Lactobacillus strains Selur 6 and Selur 19, is shown, which are sensitive to four (i.e. gentamicin, tetracycline, erythromycin and vancomycin) of the most clinically-applicable antibiotics. When the transition points of Danielsen and Wind (2003) are applied, Lb.

gasseri is just as well susceptible to these four antibiotics. According to FEEDAP Panel Report (European Commission, 2005), Str. thermophilus Selur 12 is resistant to gentamicin and erythromycin. All strains possess streptomycin resistivity according to FEEDAP (European Commission, 2005) boundary points. When the boundary points ..of Danielsen and. Wind _. (2003) were applied^ it was. fpujid out ..that Lac tobacillus . i strains were sensitive to streptomycin. Since there is no boundary point value for metronidazol, cefotaxim, cefachlor and amoxicillin, the strains cannot be defined as sensitive or resistant. High MIC values were achieved for all metronidazol strains. This results are in agreement with Delgado et al. (2007) who has noticed substantial resistivity to this antibiotic with most Lactobacillus species, when testing human gastro-intestinal tract isolates. Str. thermophilus Selur 12 is resistant to azithromycin, when the microbiological boundary point is taken into account in agreement with CLSI/NCCLS for clinical studies. No boundary point value has been suggested for this antibiotic by the FEEDAP Panel Report (European Commission, 2005).

Proving some probiotic characteristics of the new strains is a particularly favoured invention's implementation. In this respect, the new strains subject to this invention were examined for anti-microbial activity, hydrophobia, β-galactosidase activity, ability to adhere to Caco-2 cells, competitive and exclusion properties and immune reaction of the THP-1 macrophages on stimulation wuth Lb. bulgaricus strain Selur 6. " - Anti-bacterial activity was tested- y agar point test to 31 indicative strains ^; including various milk bacteria, Clostridium perfringens lb 1106, CI. tyrobutyricum DSM 2637, Bacilus cereus CCM 2010 ^T , three strains of Staphylococcus aureus, Enterococcus durans CCM 5612, two strains of Enterococcus faecalis, E. faecium LMG 11423, Listeria monocytogenes IM 372, L. innocua ATCC 33090 and E. coli 08:K88. As shown in Example 4, a wide range of anti -microbial activity was proven particularly for Lb. bulgaricus strains Selur 6 and Selur 19 (Table 8). Extensive anti-microbial activity of Lb. bulgaricus Selur 6 and Selur 19 strains was observed not only against Lactobacillus indicative strains, but also against S. aureus, Li. monocytogenes and Li. innocua. Str. thermophilus Selur 12, in addition to milk bacteria, inhibited only moderately enterococci and Listeria strains. None of the new strains inhibited Clostridium perfringens lb 1106, CI. tyrobutyricum DSM 2637, Enterococcus durans CCM 5612, or E. coli 08:K88. The anti-microbial activity of Lb. gasseri K7 against various strains of Clostridium tyrobutyricum and CI. difficile was proven earlier (Bogovii MatijaSid and Rogelj, 2000, Bogovic ^" Matijasic et al., 2007).

:.- -·... Hydrophobia was determined according to Vinderola and Reinheimer (2003) : by measuring bacterial cell separation between organic (n-hexadecane) and water phases. As described in Example 5, high cell-surface hydrophobia was established for both Lb. gasseri K7, as well as for Lb. bulgaricus Selur 19.

Highest β-galactosidase activity, determined after the protocol of Vinderola and Reinheimer (2003), using the synthetic compound o-nitrophenil-fi-D-galactoside (OPNG), was demonstrated by Lb. bulgancus strain Selur 6. Compared to the other strains, both Lb. bulgaricus strains Selur 6 and Selur 19 manifested excellent β- galactosidase activity (Example 6), which is a very good sign for all of these with decreased lactase activity.

An important functional property of probiotic bacteria is the strain's ability to adhere to intestinal epithelial cells. Our experiments used Caco-2 cells as in vitro model. The detailed procedure is described in Example 7 jointly with the competitive and exclusion properties test of Lb. bulgaricus strain Selur 19 against two strains, S. aureus ATCC 29213 and E. coli ATCC 25922. The adhesion of Lb. bulgaricus Selur 6 and Selur 19 and Str. thermophilics e\ur 12 strains was.as a whole at, least comparable or in a certain example, when the adhesion % (adhesion cfu/ axilla divided into added cfu/ axilla represented in %) was calculated, even better than that established for Lb. gasseri K7 and some other probiotic strains (Lb. rhamnosus GG, Lb. johnsonii Ljl,

Lb. reuteri ING 1, Lb. Shirota SHI). In the competitiveness analysis, Lb. bulgaricus Selur 19 was capable of inhibiting the sticking of E. coli ATCC 25922, but not that of S. aureus. In the exclusion analysis, Selur 19 was not effective.

Modulation of immune reaction is one of the health-beneficial effects of probiotic bacteria. Since some of the preliminary experiments pointed to a possible immune-stimulating effect for Lb. gasseri K7 and Lb. bulgaricus strain Selur 6, both strains were tested for their ability to influence cytokene release from a cell line of THP- microphages. On account of their well-known probiotic effects, including modulation of immune reaction, Lb. rhamnosus GG and Lb. examplei DN-114001 were used as positive control. The experiment is described in Example 8. Selur 6 and K7 strains did not cause release of inflammatory cytokenes (IL-8, IL-6 and TNF) from the m^ wmle _: b th,stra concentration of one of . ; the main inflammatory cytokenes caused by the addition of LPS (lipopolysaccharides

£. coli 0111:B4). The reduced release of inflammatory cytokines shows that Selur 6 and K7 strains could possess anti-inflammatory effect. The distinguished increase in IL-Ιβ could result in lymphocyte B proliferation and subsequent production of IgA, which should be confirmed in vivo.

Probiotic preparations consisting of at least one of the new strains on their own or

in combination with Lb. gasseri strain K7

Lactic acid bacteria as a whole are used as starter culture to produce daily foods or other types of foods. Starter cultures containing at least one of the strains, according to the invention, are another invention implementation. As a whole, such starter culture composition contains bacteria in concentrated form, including frozen, dried or lyophilised concentrates, possessing on the overall live cell concentration within the range from 10 ⁶ to 10 ¹¹ cfu (colony forming units) per gram of the composition, including at least 10 ⁶ cfu per gram of the composition, such as 10 ⁷ .... ..cfu/ g, .i.e. at leas lOLcfu/g, such as at.least .10 .cfu/g _/ .i.e..,at.least,10J° fu/.g _/J .such. as ^, .. ^, ,·--, ^, at least 10 ⁿ cfu/g.

An invention aspect is also a composition, containing each of the bacterial strains, according to the invention, jointly with Lb. gasseri K7 or another bacterial strain. In this connection, this invention refers to biologically pure cultures of each of the strains or mixtures therewith, or with Lb. gasseri K7, or with other bacterial strains. Thus, the mentioned aspect of this invention is to obtain various compositions, including probiotic ones, containing at least one strain or mixture of strains according to the invention.

When lactic acid bacteria are used as starter culture to obtain fermented food, for instance fermented milk, the knowledge of strains' fermentation activity and the combinations which provide the great number of live strains is crucial. The study of the various strain combinations and fermentation temperatures is described in

Example 9. Str. thermophilus strain Selur 12 gave evidence of a good lactic bacteria stimulator. Milk inoculated with various strain combinations fermented (an average - , pH 4,6 , was reached) in 5 to 7;5 hours. Very good development and survival of the · probiotic strain Lb. gasseri K7 during milk fermentation with Sir. thermophilus strain Selur 12 and Lb. bulgaricus strain Selur 19 was established at two fermentation temperatures, 42°C and 37°C, accordingly. The number of live cells of Lb. gasseri K7 in fermented milk reached the level of 8,70*10 ⁸ to 1,6*10 ⁹ cfu/ ml, when fermentation was carried our at 42°C and 37°C, accordingly. It was confirmed that Lb. gasseri K7 may be combined with the new strains for probiotic food products and preparations.

The next important implementation of this invention is a technology for producing biomass and probiotic starter cultures, food supplements for humans and animals, and pharmaceutical preparations. The procedure is presented in details in

Example 10. The quoted compositions may be based on the fermentation of milk, whey, or plant substrates, such as soy, rice, or other grain milk.

As a whole, a preparation based on milk fermentation, which may be used as starter culture, contains lyophilised bacteria in combination with live cells which vary between 10 ⁷ and 10 ¹⁰ cfu per gram of the preparation, including at least 10 ⁷ cfu/g, . for- instance at least 10 ⁸ cfu/g, such as at least 10 ⁹ cfu/g>-such - as at least 10 ¹⁰ · ^■ - · cfu/ g, less than 400 mg lactose per gram and lactulose which varies between 25 and 50 mg/g.

Another aspect of the invention consists in a food product containing carrier material and at least one strain, according to the invention, culture, composition, or product, according to the invention. It is preferable that the carrier material be food selected among milk, yoghurt, cheese, fermented milk, milk based on fermented products, meat based on fermented products, fermented-based-on-wheat products, powders based on milk or wheat, baby food, food supplements for humans or animals, ice creams, juices, candies, bread, cakes, or chewing gums. In preferred implementation, the microbial strain of this invention is contained at a level between about 10 ⁶ and about 10 ¹¹ cfu/ g of the carrier material, preferably between about 10 ⁶ and about 10 ⁹ cfu/ g, more preferably between about 10 ⁷ and about 10 ⁹ cfu/g of the carrier material.

In another aspect, this invention provides probiotic food supplement for human and animal feeding and pharmaceuticarcompositiohs containing at least " ' strain, according to the invention, combined with Lb. gasseri K7 only or with Lb. gasseri K7 and other nutrition substances, probiotics, minerals, vitamins, or other food or/ and pharmaceutically acceptable substances. To obtain the quoted preparations, according to this invention, at least one of the strains of this invention should be available in quantity between 10 ⁷ cfu/g and about 10 ¹² cfu/g of the carrier material and Lb. gasseri strain K7 should be available in quantity between 10 ⁸ cfu/ g and about 10 ¹² cfu/ g of the carrier material, preferably between 10 ⁹ cfu/g and about 10 ¹² cfu/g, more preferably between 10 ¹⁰ cfu/g and about 10 ¹² cfu/g of the carrier material.

Probiotic food supplements for humans and animals and pharmaceutical preparations may be obtained in the form of tablets, capsules, granules, liquid bacterial suspensions, paste or powder. In addition, the probiotic composition, according to this invention, may be any digestible material chosen from within the group consisting of milk, curds, products based on fermented milk, sour milk, -yoghu rt - frozen- =-yoghurt> milk- powder,- powders - based on ^j milk; - milk- concentrate, " cheese, cream cheeses, drinks, ice creams, products based on fermented wheat, baby foods, tablets, liquid bacterial suspensions, dried oral supplement, wet oral supplement, dry food for feeding animals through a tube or wet food for feeding through a tube which has been obtained by the use of at least one strain, according to the invention, in combination with Lb. gasseri strain K7.

In another implementation, the mentioned composition contains in addition food and/ or pharmaceutically acceptable substances and pharmaceutically acceptable carrier.

In useful implementations, the probiotic composition, according to the invention, is suitable for prevention or treatment of a disease, a syndrome, or a condition from the group consisting of antibiotic-related disorders, gastroenteritis, diarrhoea, including diarrhoea with travellers or acute child diarrhoea, lactose intolerability, gastro-intestinal infections, and development of pathogenic bacteria in the gastro-intestinal tract, inflammatory bowel disease (IBD), or other to

EXAMPLES

Example 1:

Characterization of the new strains Lb. bulgaricus and Str. thermophilus

The isolated strains were purified 3 times, according to the classical method, on M17 (Str. thermophilus Selur 12) and MRS (Lb. bulgaricus Selur 6 and Selur 19) agar, and characterised by genotyping.

. In addition to the new strains, Lactobacillus delbrueckii ssp. bulgaricus (Selur 6, and Selur 19) and Streptococcus thermophilus Selur 12, reference strains obtained from collections of microbial cultures (LMG 6901 ^T Lb. delbrueckii ssp. bulgaricus, LMG 6412 ^T Lb. delbrueckii ssp. delbrueckii), strains isolated from traditional yoghurts (milk -bacteria: L7> L8; M L12 streptococcus -bacteria: ST4-M ST7) and- strains-isolated from . a - set of various yoghurts obtained from the market were used for comparison.

The Lactobacillus strains were cultivated in MRS broth/ MRS agar, and the Streptococcus strains - in M17 broth/M17 agar (Merck, Darmstadt, Germany) at 42°C. Genome DNA was isolated from the pure cultures of the new isolates and from the reference strains using Wizard ^® set for purification of genomic DNA (Promega) or using Maxwell™ 16 system (Promega).

Type- and subtype-specific PCR

PCR with Lb. delbrueckii type-specific primers Del I (5 -ACG GAT GGA TGG AGA GCA G-3') and Del II (5'-GCA AGT TTG TTC TTT CGA ACT C-3") and Str. thermopkilus type-specific primers Th I (5'-ACG GAA TGT ACT TGA GTT TC-3') and Th Ii (5 -TTT GGC GTT TCG ACC TAA C-3') (Tilsala-Timisjarvi and Alatossava, 1997) was carried out on DNA extracted from pure cultures of selected strains for type identification. DNA fragments of expected cca. 200 bp were obtained after PCR with Lb. delbrueckii type-specific Dell and Del Il primers (Fig. 1.) and DNA fragments of expected cca. 260 bp were obtained after PCR with Str. thermopkilus type-specific Th I and Th II primers (Fig. 2). A fragment of greater size is probably a dimer and another subtype reference strain from the collection Lb. delbrueckii ssp. delbrueckii (Fig _: 1) is lacking.

The subtype identification of Lb. delbrueckii was carried out with primers LB1 (5'-AAA AAT GAA GTT GTT TAA AGT AGG TA-3') and LLB1 (5'-AAG TCT GTC CTC TGG CTG G-3 ¹ ) (Torriani et al., 1999). The DNA fragments of expected cca. 1065 bp were obtained after PCR with Lb. delbrueckii ssp. bulgaricus subtype-specific LB1 and LLB1 primers. No expansion products were observed in another subtype collection of strains Lb. delbrueckii ssp delbrueckii (Fig. 3). Identification of strains by sequencing of PCR expanded variable regions VI and V3 of 16S rPHK

To confirm additionally type identification, sequencing of the PCR expanded variable regions VI and V3 of 16S rPHK was carried out. The isolated genome D A of Lb. delbrueckii ssp. bulgaricus strains (Selur 6 and Selur 19, and L7, L8, L12), Str. thermophilus Selur 12 strain and Lb. gasseri K7 strain was PCR expanded by primers P1(5'-GCG GCG TGC CTA ATA CAT GC-3") and P4 (5'-ATC TAC GCA TTT CAC CGC TAC-3') to obtain 660 bp sized variable regions VI and V3 of 16S rPHK of lactic acid bacteria for identification and classification of the type, as described earlier by Klijn et al. (1991). The PGR protocol was modified by the heating (at 52°C for 30 s) and elongation (at 72°C for 30 s) stages. The PCR products were purified by gel electrophoresis and extracted from the gel using Wizard ^® SV Gel arid PGR cleaning system (Pf omega). Sequencing of the PGR products was carried but by Microsynth Lab in Balgach, Switzerland. The obtained sequences were then analysed directly in the NCBI data base by BLAST search to determine the homology with the nucleotide sequences frOrri me GehBa¾ data base and then, the type/subtype was determined.

Lactobacillus strains Selur 6 and Selur 19 were identical (99%) with the reference sequence of Lactobacillus delbrueckii subtype bulgaricus ATCC BAA-365 or ATCC 11842, Selur 12 strain was identical (100%) with the reference sequence of Streptococcus thermophilus L G 18311. It was re-confirmed that strai K7 was identical (100%) with the reference sequence of Lactobacillus gasseri ATCC 33323.

In agreement with the previous results obtained from PCRs with the type- and subtype-specific PCR primers (Figs. 1, 2 and 3), the new strains Selur 6 and Selur 19 were confirmed as Lactobacillus delbrueckii ssp. bulgaricus, and the strain Selur 12 - as Streptococcus thermophilus.

RAPD-PCR amplification

Several oligonucleotide primers were used individually in a series of PCR amplifications for the purpose of finding the best _v possibility to allocate RAPD characteristics among the Lb. delbrueckii ssp. bulgaricus strains and to differentiate the new strains from y.arious„Lfc. bulgaricus strains -.used for . industrial production of yoghurt:

• M13 (Torriani et al.,1999) (5'-GAG GGT GGC GGT TCT-3') (2 protocol versions were applied: protocol described by Torriani et al.(1999) - 4 mM MgCb and heating temperature of 45°C for 20 s - and protocol developed by Rossetti and Gifaffa (2005) - 3 mM MgCh and heating temperature of 42°C for 20 s),

• OPL-01 (Van Reenen and Dicks, 1996) (5'-GGC ATG ACC T-3'),

• VALIO (Tynkkynen et al., 1999) (5'-AGT CAG CCA C-3'), 125 (Torriani et al.,1999) (5'-CCG CAG CCA A -3*),

• KGT-70GC* (5--AGC GGG CGT A-3' ) and

• KGT-80GC* (5*-CGC GTG CCC A-3") with new protocol of 4mM MgCb arid cyclic programme of 95°C for 2 min, 35 cycles: 95°C for 1 min, 37°C for 2 min, 72°C for 2 min and final elongation of 72°C for 5 min).

*The oligonucleotide primers KGT-70GC and KGT-80GC were constructed by the Head of the Dairy Science research group (University of Ljubljana, Biotechnology Faculty)

Amplification products were analysed by electrophoresis in 1,5% gels with TAE buffer and coloured with SYBR Safe DNA gel colouring agent (Molecular Probes, Invitrogen) and photographed.

The most compliant in number, distribution^ intensity of shown strain differentiation bands appeared to be RAPD primers 1254 (Fig. 4) or KGT-70GC (Fig.

5)·

EXAMPLE 2:

Survival of the new strains in simulated gastro-intestinal tract (GIT) conditions

Various tests were carried out according to the FSA (2005). In addition to the strains Selur 6, Selur 19, and Selur 12, strain Lb. gasseri K7, isolated from child faeces, and. strain ./.b. bulgaricus L7, ~L8 . w L12 isolated from traditional yoghurts were included in the tests for comparison.

Survival in simulated gastric juice (SGT)

A medium simulating gastric juice (SGJ) was made of peptone water (7.5 g/1) at pH 1, 2, 3 and 6.5, using 1 M hydrochloric acid. Before the experiment, a fresh solution of pepsin (300 mg/L) was added in a quantity to reach final concentration of 15 mg/L. 10 ml of cultures cultivated overnight on the strains were concentrated by centrifugation and a second suspension in 1 ml SGJ. At time T 0 and after 20 min, 1 ml of the specimen was diluted in peptone water and sown on MRS or M17 agar. The Petri plates were incubated for 48 h at 37°C. The results are presented as a ratio (r) between the logarithmic value of the live cells after 20-minute treatment and the logarithmic value of the live cells before treatment. According to the results, at pH 1, only strains Selur 6 and Selur 12 survive at 20-minute treatment in SCJ at a rate comparable with the strain Lb. giisseri K7. At pH 2, approximately half of the live cells of all tested strains survive, as well as 100% of the Lb. gasseri K7 cells. pH 3 and 6,5 are partially fatal only for Sir. thermophilics Selur 12 (Table 1).

Table 1. Survival of Lb. bulgaricus strains L7, L8, L12, Selur 19, Selur 6, Lb. gasseri K7, and Str. thermophilus Selur 12 strains in simulated gastiic-mtestinal juice at pH 1, 2, 3 and 6,5 for 20 min (r are the mean values of both tests)

K7 ^• 0,54 0,00 1,00 ^" 0,01 0,99 ^' 0,01 0,99 ^" 0,01

Selur 12 0,00 0,00 0,55 0,01 0,86 0,03 0,96" 0,06

Key:- - r - mean for cells at T20/ mean for cells at TO

SD - standard deviation

r=l - the cells survive at 20-minute exposure to SGJ,

r>l - strain growth,

r=0,5 - loss of half of the live cells available in culture

Survival in simulated upper intestinal juice (UID

Simulated upper intestinal juice (UIJ) composed of bile salts (1 g/1) suspended in lx PBS buffer (pH 8). Before the experiment, freshly obtained solution of pancreatic enzyme (1 g/L) is added. 1 ml of culture developed overnight or 1 g of the preparation with strains was inoculated in 10 ml UIJ and incubated at 37°C for 2 hours. At T 0 and 2 h, 1 ml of the suspension was diluted and sown on MRS or M17 agar. ¾ Reduction in the number of - ^■ < ^'■ " cells in three or four logarithmic units immediately after the exposure to UIJ (npw T 0) was observed for all tested strains in the pure cultures. After 2 h treatment, additional reduction by two orders was observed only for control strain L12. Sir. t\wrmoph\lus Selur 12 was not affected immediately while the incubation for 2 hours was fatal for this strain (Table 2). When the strains' biomass was obtained by milk fermentation in bioreactor (as described in Example 10) and a lyophilised preparation with strains was exposed to UIJ, the strains! survival was better (Table 3).

Table 2. Survival of strains Lactobacillus L7, L8, LI 2, Selur 19, Selur 6, K7 and strain

Str. thermophilus Selur 12 in simulated UIJ after incubation at 37°C for 2 hours (the logio values have been averages from two tests) Strain Contents SD TO SD T2 SD (cfu/ml) -

L7 7,40 0,27 3,77 0,53 3,29 0,26

L8 8,23 0,21 4,01 0,73 2,78 _. .1,17

L12 8,70 0,08 5,41 0,46 3,76 0,20

Selur 19 8,43 0,54 5,54 0,90 5,17 0,84

Selur 6 7,85 0,42 3,92 0,71 4,03 0,12

K7 8,68 0,11 4,80 0,45 4,20 0,80

Selur 12 7,80 0,25 7,64 0 5 0,35 0,60

Key:

Contents cfu/ ml - initial number of cells (colony forming units/ ml) values

TO - log™ cells in the beginning - time 0

T2 - logio cells after 2-hour incubation _

SD - standard deviation

Table 3. Survival of Lb. bulgaricus Selur 6 with production based on milk in simulated UIJ after incubation at 37°C for 2 hours

Key:

Selur 6 cfu/ g - initial number of cells (colony forming units/ g) in logio values

TO - logio cells in the beginning - time 0

T2 - logio cells after 2-hour incubation . ........ . ,..... .. EXAMPLE 3: ... ...... .........

Profiles of the antibiotic susceptibility of the new strains

The strains' susceptibility to new antibiotics was analysed by the E-test method (AB Biodisk, Solna, Sweden) using bands to determine streptomycin (SM), gentamicin (GM), tetracycline (TC), ampycillin (AM), metronidazole (MZ), clindamycin (CM), chloramphenicol (CL), erythromycin (EM) rifampicyn (RI), vancomycin (VA), cefotaxime (CTL), cefaclor (CF), amoxicillin (AC), azithromycin

from 2 tests are shown; l.parall., 2. parall).

METRONIDAZOLE (MZ) (0,016-256) - - > 256 > 256

CLINDAMYCIN (CM) (0,016-256) 4 64 8 8 r/s

CHLORAMPHENICOL (CL) (0,016-256) 4 16 6 r/s

ERYTHROMYCIN (EM) (0,016-256) 4 1 0,19 0,19 s

RIFAMPICYN (RI) (0,016-256) - 2 0,094 0,094 s

VANCOMYCIN (V A) (0,016-256) 4 4 1,5 2 ; s

CEFOTAXIME (CTL) (0,02-32) - - 1,5 2 ^■ 7

CEFACLOR (CF) (0,016-256) - 16 16 ?

AMOXICILLIN (AC) (0,016-256) - 1 5 1,0 ?

AZITHROMYCIN (AZ), (0,016-256) _; . ;¾ i;5 - 1,5 ^{ί ÷}

CLARITHROMYCIN (CH) (0,016-256) - 8 0,125 0,125 s

AMOXICILLIN/CLAVULANATE (XL)

- 16/8 0,75 0,75 s (0,016-256)

MICbp ³ : microbiological interruption point for the mandatory homof ermentation Lactobacillus species, in accordance with the FEED AP Panel Report (European

Commission, 2005),

MICbp ^b : microbiological interruption point in accordance with Danielsen and Wind (2003).

s/r: sensitive/ resistant

- : N.A.

?: not specified, since the boundary point for this antibiotic has not been stated

Table 5. Results for MIC by Etest ^® (AB Biodisk, Sweden) for Lb. bulgancus Selur 6 (the results from 2 tests are shown; l.parall., 2. parall ). MlC ^g/ml)

Lactobacillus bulgaricus Selur 6 s/r

MICbp ³ MICbp ^b 1. par all. 2. parall.

STREPTOMYCIN (SM) (0,064-1024) 16 > 256 32 .48 r/s

GENTAMICIN (GM) (0,064-1024) 8 256 6 6 s

TETRACYCLINE (TC) (0,016-256) 8 4 1,5 1,5 s

^' AMPYCILLIN (AM) (0,016-256) 4 4 0,125 0,125 s

METRONIDAZOLE (MZ) (0,016-256) - - 256 > 256 ?

. CLIND AMY CIN (CM) (0,016-256) 4 64 0,064 0,064 s

.CHLORAMPHENICOL (CL) (0,016-256) ,4 16 3 s

ERYTHROMYCIN (EM) (0,016-256) 4 1 0,047 0,047 s

^" RIFAMPICYN (RI) (0,016-256) - ^■ 2 0,38 0,38 s

VANCOMYCIN (VA) (0,016-256) " ^•• 4" ^{··'· '·""" '} 4 " 1,0 1,0 s

CEFOTAXIME (CTL) (0,02-32) - - 0,094 0,094 ?

CEFACLOR (CF) (0,016-256) - - 1 1 ?

AMOXICILLIN (AC) (0,016-256) - - 0,064 0,064 ?

AZITHROMYCIN (AZ) (0,016-256) - 8 0,25 0,19 s

CLARITHROMYCIN (CH) (0,016-256) - 8 ; 0,064 0,047 s

AMOXICILLIN/ CLAVULANATE (XL) - 16/8 0,064 0,064 s (0,016-256)

MICbp ³ : microbiological interruption point for the mandatory homofermentation Lactobacillus species, in accordance with the FEEDAP Panel Report (European

Commission, 2005).

MICbp ^b : microbiological interruption point in accordance with Danielsen and Wind (2003). s/r: sensitive/ resistant - : N.A.

?: not specified, since the boundary point for this antibiotic has not been stated

Table¾. Results for MIC by Etest ^® (AB Biodisk, Sweden) for Lb. bulgaricus Selur 19 (the results from 2 tests are shown; l.parall., 2. parall.).

MICbp ³ : microbiological interruption point for the mandatory homofermentation Lactobacillus species, in accordance with the FEEDAP Panel Report (European Commission, 2005). - MIC _b p : microbiological interruption point in accordance, with Danielsen and Wind (2003).

s/r. sensitive/ resistant

- : N.A.

?: not specified, since the boundary point for this antibiotic has not been stated fable 7. Results for MIC by Etest ^® (AB Biodisk, Sweden) for Lb. bulgaricus Selur 12 (the results from 2 tests are shown; l.paralL, 2. parall.).

AZITHROMYCIN (AZ) (0,016-256) ^{" "'} 8 32 2 " _r

CLARITHROMYCIN (CH) (0,016-256) - 8 6 6 s

AMOXICILLIN / CL A VUL AN ATE (XL) - 16/8 0,19 0,125 s (0,016-256)

MICbp ^a : microbiological interruption point for the mandatory homofermentation Lactobacillus species, in accordance with the FEEDAP Panel Report (European

Commission, 2005).

MICbp ^b : microbiological interruption point in accordance with Danielsen and Wind (2003).

s/r: sensitive/ resistant

- : N.A.

?: not specified, since the boundary point for this antibiotic has not bee stated

EXAMPLE 4:

Antibacterial activity of the new strains

The antibacterial activity of all strains was tested against 31 indicative strains, including various lactic acid bacteria, Clostridium perfringens lb 1106, CI. tyrobutyricum DSM 2637, Bacilus cereus CCM 2010 ^T , three strains of Staphyilococcus aureus, Enterococcus durans CCM 5612, two strains of Enterococcus faecalis, E. faecium LMG 11423, Listeria monocytogenes IM 372, L. innocua ATCC 33090 and E. coli 08:K88.

Agar-spot test was conducted, using MRS agar with decreased (0,2% wt/v) glucose concentration for the strains Lactobacillus and M17 agar for the strain Str. thermophilus Selur 12 and 24-hour incubation of the tested bacterial cultures before covering the plates with indicative microorganisms. The covering agar was obtained from 5 ml of the respective soft agar for various indicative strains (MRS for lactic acid bacteria, BHI for B. cereus, E. coli, S. aureus and Listeria strains, RCM for Clostridium strains and M17 for enterococci) and 50 μΐ of 18-h indicative cultures. Subsequent 24- h incubation of the covered plates was carries out in conditions appropriate for the individual indicative strains (30°C/aerobiosis for B. cereus and Lb. sakei, 37°C/ aerobiosis for lactic. acid. bacteria, the enterococci E. colt, .S. aureus and Listeria strains, , ... , 37°C/ anerobiosis for Clostridia).

Only the results for the indicative strains incubated from at least one tested strain are presented on Table 8. Fig. 6 shows the anti-microbial activity of Lb. bularicus strains Selur 6 and Selur 19 and Lb. gasseri K7 strain against Staphylococcus aureus ATCC 29213.

Table Anti-microbial activity of the new strains (Lb. bulgaricus Selur 6 and Selur 19, Sir. thermophilus Selur 12) and the Lb. gasseri K7 strain

S: aureus IM 388 ^{" '" ' " '} +++ ^{"" " '} +++ ^{" ' "**} +++

S. aureus IM 390 +++ +++ (s) +++

£. coli ATCC 25922 - + ++ n.d.

E. coli ATCC 11229 - - + n.d.

Li. monocytogenes IM 372 +++ +++ +++ ■ +

Li. innocua ATCC 33090 +++ +++ +++ +

Key:

*: point test was carried out on MRS agar plate

² : point test was carried out on M17 agar plate

-: no inhibition area was observed

+: inhibition area varying between 0 and 2 mm

++: inhibition area varying between 2,5 and 5mm

+++: inhibitiort area varying between > 5,5 mm

s: acute-angle area which is usually caused not only by acid, but by other anti^ - microbial substances as well

n.d.: not defined

EXAMPLE 5:

Hydrophobic properties of the new strains

Hydrophobia was determined according to Vinderola and Reinheimer (2003) by measuring the separation from bacterial cells between the organic (n-hexadecane) and water phases. The hydrophobia of the cellular surface (%H) was then calculated using the following formula:

%H = (Ao-A)/ Ao*100 where Ao and A are the optic densities before and after extraction with organic diluent, accordingly. On addition to the strains Lb. bulgaricus Selur 6 and Selur 19, the -strains Lb. bulgaricus L7, L8 and L12, isolated from traditional Bulgarian yoghurts and Lb. gasseri K7 were included in the testing for compariscioci. .... . , .High, surface-cell , hydrophobia was established for Lb. gasseri JK7 .and Lb. bulgaricus Selur 19. The hydrophobia of all other strains was quite low and varied between 12.8 and 16.9 % (Table 9).

- Table 9. Hydrophobia of the new strains Lb. bulgaricus and Str. thermophilus

Example 6: β-galactosidase activity of the strains β-galactosidase (β-Gal) activity of the new strains was studied after the protocol of Vinderola and Reinheimer (2003), using the synthetic compound o- nitrophenyl-fi-D-galactopyranoside (ONPG). The function of β-Gal in the cell is to hydrolyse lactose to glucose and galactose so that they might be used as a carbon source. The synthetic compound OPNG is a substrate which, when hydrolyzed, produces galactose and o-nitrophenol, which has yellow colour. When OPNG is in excess with respect to the enzyme in the reaction, the production of o-nitrophenol per unit time is proportional to the concentration of β-galactosidase; thus, yellow colouration may be used to determine enzyme concentration. The absorption values at 420 and 560' nm were determined for each specimen and ^β -Gal activity was calculated (in Muller units = standardized quantity of β-Gal activity), as follows:

1000 x ^{( 420 5 x AZ ))} _{z Mu} u _{er units}

(tx vx A\ ₅₆₀ ) t = reaction time in minutes

v = culture volume analysed in millilitres

Λ420 = absorption of yellow o-nitrophenol

A2s ₆ o = absorption value of the reaction mixture (dissipation from cellular residues which, when multiplied by 1,75, approximates the dissipation observed at 420 nm)

AI560 = refers to the cellular density in washed-out cellular suspension before analysis

For the comparison, three well-known probiotic strains were included in the test (Lb. rhamnosus ATCC 53103, Lb. examplei DN-11401 and Lb. examplei Shirota) (Table 10).

Table 10. fi-galactosidase activity of the new strains

·.· - ,.· -..,. ..·- ... ..... ... Example,?:.

Adhesion ability of the new strains

An important functional property of probiotic bacteria is the strain's ability to adhere to epithelial cells. Our experiments used Caco-2 cells as in vitro model. Adherence studies contain the following stages: a) Caco-2 human colon adenocarcinoma cell line (IZSBS BS TCL 87) was routinely cultivated in Eagle environment modified after Dulbecco (DMEM) (Sigma, CAIH) with L-glutamine arid 10 % (volume fraction) from calf foetus serum Fetalclone II (Hyclone, Germany) and 50 μg/ iriL of gentamicin sulphate (Sigma, USA). Incubation was carried out at 37°C in 10% CO2 atmosphere. b) To analyse adhesion, the Caco-2 cells were sown at cell concentration of 10 ⁴ cells/ axilla in 24-axillae standard tissue culture plates. The cells were retained for 32 weeks after mergifig, when they were considered as completely differentiated. At least 24 h before the tests, DMEM environment with gentamicin was replaced by the same environment without antibiotic. Lactic acid arid streptococci cells from 18-h MRS cultures were obtained by centrifugation (3500 g/10 iriiri) and washed-out once with PBS buffer (pH=7.3) and once with a buffer used in the analysis (DMEM without FBS and antibiotic, pH=7). Cellular density was brought to optic density of 0.5 by measuring absorption at 600 ran. Bacterial cells were deposited by centrifugation and then re-suspended in half volume of DMEM, pH=7. The exact number of live bacterial cells used in the analyses was determined for each experiment by counting the colonies on MRS (for lactobacilli) or Ml 7 (for streptococci) agar. c) Adhesion analyses were carried out in 24-axillae standard tissue culture plates.

After washing-out, the Caco-2 monolayer was added twice to each axilla with PBS (pH=7.3), 0.5 mL of lactobacilli or streptococci suspension un DMEM (containing the cells from 1 ml of lactobacilli suspension with OD=0,5) and

, „.. .Mixtures of lised Caco-2.cells.and_bacterial cells, were placed on MRS agar (Merck,

Germany). Counting was carried out after 72 h incubation at 37°C in microaerophillic atmosphere. Each bacterial strain was tested in ten axillae. The results are the mean value of 10 data and are presented in Fig. 7.

Lb. bulgaricus Selur 19 was also tested for competition and exclusion properties against the two strains, S. aureus ATCC 29213 and E. coli ATCC 25922. The last two strains were chosen based on the results from agar point analyses - they were inhibited by Lb. bulgaricus Selur 19. S. aureus ATCC 29213 and E. coli ATCC 25922 were cultivated in BHI broth and produced, as described for lactobacilli, with the only difference that 10-fold more diluted cell suspensions than those of lactobacilli were applied in the test. (The suspensions were regulated up to OD-0,5, subsequent dilution to 1:10, centrifuged; then, the cells were re-suspended in appropriate quantity of DMEM). " "

In the competition analysis (Lb. + S. aureus; Lb. .+ E. coli), the lactobacilli were added to the Caco-2 cells in the axilla simultaneously with S. aureus and E. coli. To test the possible exclusion of S. aureus w E. coli by the lactobacilli (Lb. / S. aureus; Lb. / E. coli), the incubation of Caco-2 cells with lactobacilli was followed by washing-out of the unrelated cells, addition of S. aureus or E. coli and subsequent 30 rnin incubation. The Caco-2 cells were lised by 0.05% Triton X-100 (Sigma Chemical Co., St. Louis, MO, USA) for 5 min, and the stuck S. aureus and E. coli were counted (cfu/ axilla) by counting the plate at BHI and VRB agar environment (Merck), accordingly. The BHI and VRB plates were incubated for 24 h at 30°C.

The adhesion of Lb. bulgaricus Selur 6 and Selur|19 and Str. thermophilus Selur 12 strains was, as a whole, comparable to, or in the example where the adhesion % was calculated (adhesion cfu/ axilla divided by the added cfu/ axilla, represented in %) - even better than the determined for Lb. gasseri K7 and some other probiotic strains (Lb. rhamnosus GG, Lb. johnsonii Ljl, Lb. reuteri ING 1, Lb. Shirota SHI) (Fig. 7).

When the competition between E. coli ATCC 25922 and Lb. bulgaricus Selur 19 or between S. aureus ATCC 29213 and Lb. bulgaricus Selur 19 for adhesion to Caco-2 cells was tested 1.75 x 10 ⁷ cfu/axilla from Selur 19 ancl 2.22 x lO ⁷ cfu/axilla from E. „ ₌ „„co/z ^' ,-(the ratio, of -the added cfu. E.^li: SelurA9. ,l,3- : l) or 1.75 x 10 ⁷ cfu/ axilla from

Selur 19 and 0.52 x 10 ⁷ cfu/ axilla from S. aureus (the ratio of the added cfu S. aureus: Selur 19 = 0,3 : 1) was added.

Lb. bulgaricus Selur 19 was capable to inhibit the adhesion of E. coli ATCC 25922 when both strains were added to the Caco-2 cells simultaneously at close concentrations In the exclusion analysis (Selur 19/ E. coli), the adhesion of £. coli was not inhibited. However, Selur 19 was not capable to inhibit the adhesion of S. aureus ATCG 29213, notwithstanding that lactobacilli concentration was about 3 times higher (Table 11).

Table 11. Effect of Lb. bulgaricus Selur 19 on the adhesion ability of E.coli ATCC 25922 and S. aureus ATCC 29213

* Adhesion inhibition (%) was calculated comparing the results (stuck cfu from E. coli I axilla) obtained in the analysis E. coli + Selur 19 w Selur 19/ E. coli with the adhesion of E. coli on its own (100 %).

Adhesion even better than when on its own. .,„...,, .... ., ,>,...._ ., . Example 8: . .>.·.·.-., .. ..... .

Immune response of cell line THP-1 microphages at in vitro stimulation with

Selur 6 and K7

. .. Selur 6 and K7 were tested for their ability to affect the release of cytokines of macrophages cell line THP-1. On account of their well-known probiotic effects, including modulation of immune reaction, Lb. rh mnosus GG and Lb. examplei DN- 114001 were used as positive control.

The procedure is the following:

Cell line THP-1 was stimulated by TPA (phorbol esther) for 24 hours to cause division of mature macrophages. The di ided cells were washed-out and incubated for 24 hours in fresh environment (RPMI-1640) without TPA, supplemented by 10% ox serum and antibiotics. The experiment was conducted on the third day when lactobacilli, LPS (lipopolysaccharides of E. coli Olll:B4) were added and incubated with macrophages for 24 hours. The concentration of six different cy tokenes (IL-8, IL- 1β, IL-6, IL-10, TNF in IL-12p70) was measured using BD™ CBA (Cytometric Bead Array) human inflammation kit, using liquid cytometer (BD FACSCalibur).

Selur 6 and K7 strains did not cause separation of inflammatory cytokines (IL-8, IL-6 n TNF) from the macrophage cell line THP-1, while their separation was caused by addition of LPS. When LPS and Selur 6 or K7 strains were applied simultaneously, both strains reduced the concentration of the main part of inflammatory cytokines. The most remarkable changes in cytokine concentrations were observed at the levels of H 8 (Table 12). When Selur 6 or K7 were added individually, slight increase in IL-Ιβ (mediator of local inflammation) concentrations was observed, however, the effect was not statistically significant. All immune reactions were comparable to the control strains. The concentrations of IL-10 and IL- 12p70 were not affected by any of the tested strains. Table 12. Reduction of ILT8 production caused b LPS , ,-: ,.

* strain isolated from pig intestine

Example 9:

Fermentative ability of various strain combinations

Various strain combinations and fermentation temperatures were tested. One-night pure cultures were combined and inoculated in quantity of 2% in sterile defatted milk and.incubated at 42°G and 37°C (only combinations S20 + L89 w S20 + L89 + K7). During fermentation, pH was measured and fermentation was stopped on reaching pH = 4,6. After fermentation, fermented milk specimens were sown on MRS, M17 w MRS with clindamycin (0,1 mg/1) agar for selective counting of streptococci, lactobacilli, and Lb. gassen K7.

The changes in pH values during the fermentation of the milk incubated with Str. thermophilus Selur 12 and various lactobacilli strains (Selur 6, Selur 19 and Selur 19 + k7) at ratio 1:1 or 1:1:1 (Selur 12 + Selur 19i + K7) and at temperature of 42°C and 37°C (only Selur 12 + Selur 19 and Selur 12 + Selur 19 + K7) are shown in Fig. 8. The average number (in cfu/ml) of the individual strains after fermentation is presented on Table 13. Table 13. Average number of Str. thermophilus Selur 12, Lb.bulgaricus, Selur 6 <>r Selur 19 and Lb. gasseri K7 in fermented milk specimens (cfu/ mL)

* fermentation at 37°C

Example 10:

Technology for the production of biomass and prdbiotic starter cultures, food supplements and pharmaceutical preparations

The strains are prepared as pure cultures by cultivation in the course of 18 hours (the cultivation conditions are described in Table 14). One-night broth cultures are centrifuged (10 min, 6000 g), the supernatant is removed and the cells are re- suspended in a 10 times lesser quantity in Ringer solution. Cell concentration in 1 ml of cell suspension is about lxlO ⁹ - lxlO ¹⁰ cells. Cell suspension of Selur 12, Selur 6 i

and Selur 19 are inoculated separately or in combinations in sterilized restored defatted dry milk (1% inoculum), and Lb. gasseri K7 (1% inoculum) in restored defatted dry milk supplemented with 0,4% yeast extract (Biolife 412220; addition to the milk before -milk sterilization), accordingly, to produce mother cultures, which may be used as inoculum for the fermentation in bioreactor. Cultivation takes place overnight at 42°C for Selur 12,

Biomass.was-obtained b the. fermentation of milk, whey, or plant substrates . , ... such as soy, rice or other grain milk. The stages in the procedure where milk is used as substrate are as follows: a) To create the medium, 100 g/L of defatted dry milk and 4 g/L of yeast extract are

diluted in potable water and the mixture is sterilised for 30 min in bioreactor at 115°C. After sterilization, the medium is Cooled down to temperature appropriate for the various strains or strain combinations. b) Creating biomass by fermentation - technological parameters

Example 1

The medium created in bioreactor is inoculated with 1% fresh 18-hour culture of Sir. thertnophilus Selur 12 and 1% fresh 18-hour culture of Lb. bulgaricus Selur 6 and/or Lb. bulgaricus Selur 19. Cultivation is carried out for 1 h at 42°C, after which temperature is increased to 44°C and during fermentation in the next 3-4 hours, neutralization with NH4OH is carried out three times to retain pM between 4.7 and 5.7. The medium is stirred only during neutralization. In the end of fermentation, 10% (vol/ vol) cryo-protector (200 g/L solution of sterile defatted powder milk) and the fermentation medium is cooled down quickly to 22°C and retained at this temperature for 30 min. Then, the culture is fed for liophylization.

Example 2

The medium created in bioreactor is inoculated with 2% fresh 18-hour culture of Lb. gOsseri K7 and 16-hour fermentation is carried out at 37°C without pH regulation. In the end of fermentation, 10% (vol/vol) of cryo-protector (200 g/L solution of sterile defatted powder milk) is added and the fermentation medium in the bioreactor is cooled down to 22°C for 30 min. Then, the fermentation medium is lyophilised.

Example 3

The medium created in bioreactor was inoculated with 2% fresh 18-hour culture of Lb. gasseri K7 and fermentation was carried out at 37°C without pH regulation. After 16-hour fermentation/ cultivation at 37°C, pH was increased to 5,7 .the fermentation brotruwas. inoculated with 2% fresh 18 _^ hpur culture of Lb. bulgaricus Selur 6 or Selur 19. After the sowing, the temperature was increased to 44°C and during fermentation for the next 3-4 hours, neutralization with NH4OH of pH 5,0 to pH 5,7 was carried out three times. The fermentation medium was stirred only during neutralization. In the end of fermentation, 10% (vol/vol) from cryo-proteetor (200 g/L solution of sterilized defatted dry. milk) was added and the fermentation broth in the bioreactor was cooled down to 22°C for 30 min. a) Lyophilisation - technological parameters

Depth of liquid layer - 10 mm; cooling down at 4°C for 3 hours; freezing at 40°C for 90 minutes; heating at 35°C until the end of evaporation (<6 % water).

The -preparations contain dried, lyophilised bacteria with concentration of live cells, which is usually within the range between lO ⁷ and 10 ¹ ? ef/ g, less than 400 mg of lactose /g or lactulose/g, which is within the range between 25 and 50 mg/g.

Table 14. Opturtal conditions for strain growth

Example of a product (pharmaceutical composition):. _^ ..... :. ■<

1. The required amounts of each component are extended, as a percent of total mixture amount, required to produce the batch.

2. All components are sifted through a sift with openings size of 0.5 mm:

3. Mixed:

3.1. Feeding to the mixer for 30-min mixing, but for the magnesium stearate;

3.2. Adding to the mixture from point 3.2. the preliminary sifter amount of magnesium stearate and mixing for another 10 min.

j

4. The ready-made mixture packed in containers.

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