EP2935600A2 - Process for producing conjugated linolenic acid from linolenic acid employing bifidobacterium breve, bifidobacterium bifidum, or lactobacillus oris strains - Google Patents
Process for producing conjugated linolenic acid from linolenic acid employing bifidobacterium breve, bifidobacterium bifidum, or lactobacillus oris strainsInfo
- Publication number
- EP2935600A2 EP2935600A2 EP13817928.8A EP13817928A EP2935600A2 EP 2935600 A2 EP2935600 A2 EP 2935600A2 EP 13817928 A EP13817928 A EP 13817928A EP 2935600 A2 EP2935600 A2 EP 2935600A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- cect
- strain
- strains
- depositor
- mutant
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
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Classifications
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
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- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
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- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
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- A—HUMAN NECESSITIES
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- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/51—Bifidobacterium
- A23V2400/519—Breve
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- C—CHEMISTRY; METALLURGY
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- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/225—Lactobacillus
Definitions
- a microbiological method for obtaining conjugated linolenic acid from linolenic acid, and bacterial strains for performing the method is provided.
- the present invention relates to a method for obtaining or preparing isomers of the conjugated linolenic acid (CLNA), and to lactic acid bacteria and bifidobacteria for carrying out said method.
- CLNA conjugated linolenic acid
- C18:3, c/s-9 c/s-12 c/s-15, LNA conjugated double bonds.
- isomers some of them being present in seed oil, such as C18:3 cis-9, trans ⁇ 1 , c/ ' s-13, and other being generated by biohydrogenation with ruminal bacteria, which can be found in milk and meat (as rumelenic acid C18:3 cis 9, trans 1 1 , cis 15 and isorumelenic acid C18:3 cis 9, trans 13, cis 15).
- LNA Bacterial production of CLNA from LNA is critical because LNA is considered a toxic for some bacterial cells, in such a way that the growth of the cell culture is retarded. Thus, it is difficult to obtain great amounts of CLNA and with high yields from bacteria. Indeed, bacterial transformation of LNA to CLNA is a defense mechanism against toxic agents.
- strain Bifidobacterium breve DPC6330 disclosed in Hennessy et al., "The
- compositions containing CLNA to organisms, by means of functional foods, dairy products (milk derived products), nutritional
- compositions or pharmaceutical compositions represents an interesting challenge giving raise to efficient added-value products.
- the strain can produce about 0.3 mg/ml of CLNA in a medium comprising initially 0.5 mg/ml of LNA.
- the data depicted do not indicate the amount of strain used in the assays making difficult establishing a growing kinetics.
- compositions with high amounts of CLNA as well as of compositions comprising the precursor ingredients for finally obtaining in situ effective amounts of CLNA.
- LAB Lactic Acid Bacteria
- Bifidobacteria selected from the species of the group consisting of Bifidobacterium breve, Bifidobacterium bifidum, and Lactobacillus oris
- LNA linolenic acid
- CLNA conjugated linolenic acid
- the inventors provide also a method for the production of CLNA from LNA with high conversion rates, in which the specific lactic acid bacteria and bifidobacteria are used to promote such conversion.
- the isolated strains of lactic acid bacteria and bifidobacteria are able to promote the conversion in different media, including food matrices, such as food products, or nutritional formulas, in particular in milk-based nutritional formulas, as well as in other type of milk-based compositions.
- a medium which can be an edible composition comprising at least the fat portion (from animal or vegetal origin) contained in a milk-based product, said milk-based product from animal or vegetal origin, such as a yogurt, a fermented milk, an infant formula or food supplement could be enriched with CLNA and provides thus the beneficial effects associated to this compound.
- the invention aims a method for preparing an isomer of conjugated linolenic acid (CLNA) from linolenic acid (LNA) in a medium comprising at least the fat portion, from animal or vegetal origin, contained in a milk-based product, said fat portion containing linolenic acid, the method comprising adding to the medium at least one strain used in dairy or agroalimentary industry, selected from the group consisting of a lactic acid bacteria (LAB) and bifidobacteria, said strains characterized by: i) being able to grow to an optical density of at least 0.9 OD 6 oo
- the strains usable in the method of the invention do not grow to and optical density of 0.39 OD600 (24 h in anaerobiosis).
- the strains of the invention when cultured in anaerobiosis for 24 h, grew to an optical density (OD600 nm) comprised from 0.50 to 6.00 in MRS medium, and from 0.50 to 5.00 in a medium comprising linoleic acid or linolenic acid.
- OD600 nm optical density comprised from 0.50 to 6.00 in MRS medium, and from 0.50 to 5.00 in a medium comprising linoleic acid or linolenic acid.
- the growth of the strains usable in the method of the invention was of at least 0.50 in any media.
- the strains usable in the method of the invention are those complying with the conditions (i) and (ii) above, with the proviso that said strains are also able to grow to an optical density of at least 0.5 OD600 at 24 hours in anaerobiosis either in MRS medium or in a medium supplemented with comprising linoleic acid or linolenic acid.
- strains have the advantage that can grow in many media comprising linolenic and/or linoleic acids, which are common ingredients in dietary compositions
- the method provides a conversion rate in percentage by weight of linolenic acid to conjugated linolenic acid comprised from 30 % (w/w) to 100 % (w/w), said conversion ratio calculated with the following formula:
- % of CLNA weight of CLNA / (weight of CLNA +weight of LNA) x 100.
- Another aspect of the invention is a bifidobacteria strain, which is a strain of Bifidobacterium breve deposited by the Depositor in the Coleccion Espahola de Cultivos Tipo (CECT) under the Depositor's reference ORD0123, and which received the CECT accession number CECT 8241 , or a mutant or variant thereof.
- CECT Coleccion Espahola de Cultivos Tipo
- the strain of Bifidobacterium breve of the invention isolated from infant (0-3 months) faeces from Spain was deposited, according to the Budapest Treaty, on the 29th November 2012 in the Coleccion Espahola de Cultivos Tipo (CECT) in the Universidad de Valencia CP 46980 Catedratico Agustin Escardino N° 9 Paterna, Valencia (Spain) (former in the Universidad de Valencia CP 46100 Burjasot, Valencia (Spain)), by the depositor
- Another aspect of the invention is a bifidobacteria strain, which is a strain of Bifidobacterium breve deposited in the Coleccion Espahola de Cultivos Tipo (CECT) under the Depositor's reference ORD0124, and which received the CECT accession number CECT 8242, or a mutant or variant thereof.
- CECT Coleccion Espahola de Cultivos Tipo
- the strain of Bifidobacterium breve of the invention isolated from infant (0-3 months) faeces from Spain was deposited, according to the Budapest Treaty, on the 29th November 2012 in the Coleccion Espahola de Cultivos Tipo (CECT) in the Universidad de Valencia CP 46980 Catedratico Agustin Escardino N° 9 Paterna, Valencia (Spain) (former in the Universidad de Valencia CP 46100 Burjasot, Valencia (Spain)), by the depositor
- Bifidobacterium breve was identified by the depositor with the reference ORD0124, and received the provisional and definitive CECT accession number CECT 8242.
- Another aspect of the invention is a bifidobacteria strain, which is a strain of
- the strain of Bifidobacterium breve of the invention isolated from infant (0-3 months) faeces from Spain was deposited, according to the Budapest Treaty, on the 29th November 2012 in the Coleccion Espahola de Cultivos Tipo (CECT) in the Universidad de Valencia CP 46980 Catedratico Agustin Escardino N° 9 Paterna, Valencia (Spain) (former in the Universidad de Valencia CP 46100 Burjasot, Valencia (Spain)), by the depositor
- the invention also aims a bifidobacteria strain, which is a strain of
- the strain of Bifidobacterium breve of the invention isolated from infant (0-3 months ) faeces from Spain was deposited, according to the Budapest Treaty, on the 29th November 2012 in the Coleccion Espahola de Cultivos Tipo (CECT) in the Universidad de Valencia CP 46980 Catedratico Agustin Escardino N° 9 Paterna, Valencia (Spain) (former in the Universidad de Valencia CP 46100 Burjasot, Valencia (Spain)), by the depositor
- the strain of Bifidobacterium breve was identified by the depositor with the reference ORD0134, and received the provisional and definitive CECT accession number CECT 8243.
- Another aspect of the invention is a bifidobacteria strain, which is a strain of Bifidobacterium breve deposited in the Coleccion Espahola de Cultivos Tipo (CECT) under the Depositor's reference ORD0138, and which received the CECT accession number CECT 8244, or a mutant or variant thereof.
- the strain of Bifidobacterium breve of the invention isolated from infant (0-3 months) faeces from Spain was deposited, according to the Budapest Treaty, on the 29th November 2012 in the Coleccion Espahola de Cultivos Tipo (CECT) in the Universidad de Valencia CP 46980 Catedratico Agustin Escardino N° 9 Paterna, Valencia (Spain) (former in the Universidad de Valencia CP 46100 Burjasot, Valencia (Spain)), by the depositor
- Another aspect of the invention is a bifidobacteria strain, which is a strain of Bifidobacterium breve deposited in the Coleccion Espahola de Cultivos Tipo (CECT) under the Depositor's reference ORD0294, and which received the CECT accession number CECT 8246, or a mutant or variant thereof.
- CECT Coleccion Espahola de Cultivos Tipo
- the strain of Bifidobacterium breve of the invention isolated from infant (0-3 months) faeces from Spain was deposited, according to the Budapest Treaty, on the 29th November 2012 in the Coleccion Espahola de Cultivos Tipo (CECT) in the Universidad de Valencia CP 46980 Catedratico Agustin Escardino N° 9 Paterna, Valencia (Spain) (former in the Universidad de Valencia CP 46100 Burjasot, Valencia (Spain)), by the depositor
- the strain of Bifidobacterium breve was identified by the depositor with the reference ORD0294, and received the provisional and definitive CECT accession number CECT 8246.
- Another aspect of the invention is a bifidobacteria strain, which is a strain of Bifidobacterium bifidum deposited in the Coleccion Espahola de Cultivos Tipo (CECT) under the Depositor's reference ORD0202, and which received the CECT accession number CECT 8245, or a mutant or variant thereof.
- the strain of Bifidobacterium bifidum of the invention isolated from infant (0-3 months) faeces from Spain was deposited, according to the Budapest Treaty, on the 29th November 2012 in the Coleccion Espahola de Cultivos Tipo (CECT) in the Universidad de Valencia CP 46980 Catedratico Agustin Escardino N° 9 Paterna, Valencia (Spain) (former in the Universidad de Valencia CP 46100 Burjasot, Valencia (Spain)), by the depositor
- Lactic acid bacteria strain which is a strain of Lactobacillus oris deposited in the Coleccion Espahola de Cultivos Tipo (CECT) under the Depositor's reference ORD0255, and which received the CECT accession number CECT 8240, or a mutant or variant thereof.
- the strain of Lactobacillus oris of the invention isolated from breastmilk (from a Spanish woman) was deposited, according to the Budapest Treaty, on the 29th November 2012 in the Coleccion Espahola de Cultivos Tipo (CECT) in the Universidad de Valencia CP 46980 Catedratico Agustin Escardino N° 9 Paterna, Valencia (Spain) (former in the Universidad de Valencia CP 46100 Burjasot, Valencia (Spain)), by the depositor Laboratorios ORDESA, S.L. (C/ Osca, 18-20, 08830 Sant Boi de Llobregat- Spain).
- the strain of Lactobacillus oris was identified by the depositor with the reference ORD0255, and received the provisional and definitive CECT accession number CECT 8240.
- Bifidobacterium bifidum ORD0202 (CECT 8245), Bifidobacterium breve ORD0123 (CECT 8241 ), Bifidobacterium breve ORD0124 (CECT 8242), Bifidobacterium breve ORD0128 (CECT 8239), Bifidobacterium breve
- ORD0134 (CECT 8243), Bifidobacterium breve ORD0138 (CECT 8244), Bifidobacterium breve ORD0294 (CECT 8246) were isolated from baby faeces (from Spain origin) and Lactobacillus oris ORD0255 (CECT 8240) from human breast milk (from Spain origin).
- the taxonomic characterization was performed as indicated in the Examples, namely by sequencing almost full sequence of the 16S rRNA.
- the invention also includes mutants or variants of these strains, wherein the mutants or variants of said strains are obtained using one of the deposited strains as starting material, and wherein the mutant strains retain the essential properties of the deposited strains, wherein said essential properties are: i) being able to grow to an optical density of at least 0.9 OD 6 oo
- both media comprising at least 0.3 mg/ml of linolenic acid in the medium, and ii) being able to produce conjugated linolenic acid (CLNA) in both media.
- CLNA conjugated linolenic acid
- Another aspect of the invention is a bacterial pure culture obtained from any of the strains as defined above. These pure cultures are obtained using the standard microbiological processes.
- Yet another aspect of the invention is a food product which comprises an effective amount (also named nutritionally effective amount) of at least one of the strains of the invention, together with appropriate amounts of other edible ingredients, and LNA.
- the invention in another aspect, relates to a nutritional composition which comprises a nutritionally effective amount of at least one of the strains of the invention as defined above, and LNA, together with appropriate amounts of other appropriate edible ingredients.
- the invention in another aspect, relates to a pharmaceutical composition which comprises a therapeutically effective amount of at least one of the strains as defined above, and LNA, together with appropriate amounts of pharmaceutical acceptable excipients and/or carriers.
- Another aspect of the invention relates to any of the strains as defined above, for use as a probiotic.
- a further aspect of the invention relates to any of the strains as defined above, for use as anti-obesity agent.
- This aspect can be formulated as the use of any of the strains of invention, for the manufacture of a medicament, a food product or a nutritional composition for the treatment and/or prevention of obesity in an animal including a human.
- the present invention also relates to a method for the treatment or prevention of obesity, comprising administering a therapeutically effective amount of any of the strains as defined above, or a pharmaceutical composition, or a food product or a nutritional composition comprising at least any of the strains, together with pharmaceutically acceptable excipients or carriers, or edible ingredients in a subject in need thereof, including a human.
- Another aspect of the invention is the use of the LAB and Bifidobacteria strains of the invention for the production of vitamins in a medium comprising at least the fat portion (from animal or vegetal origin) contained in a milk- based product, said fat portion comprising linolenic acid.
- the invention relates to any of the strains as defined above, for use as immunomodulator agent.
- This final aspect can be formulated as the use of any of the strains of invention, for the manufacture of a medicament, a food product or a nutritional composition for the therapeutic and/or prevention of immune system diseases of an animal including a human.
- the invention may alternatively be
- FIG. 1 is a bar diagram showing in the Y-axis the percentage of conversion (conversion rate, %) of LNA to CLNA in a medium comprising a reconstituted skimmed milk as milk-based product.
- the number under the bars (X-axis) relate to the inoculated strains Lactobacillus oris
- Bifidobacterium breve ORD0123 (identified as 123), Bifidobacterium breve ORD0124 (identified as 124), Bifidobacterium breve ORD0128 (identified as 128), Bifidobacterium breve ORD0134
- Bifidobacterium breve ORD0138 (identified as 134), Bifidobacterium breve ORD0138 (identified 138),
- FIG. 2 is another bar diagram, in which it is indicated the percentage by weight (% in the Y-axis) of the isomer C18:3 c/s-9 trans- 1 c/s-15 of the conjugated linolenic acid obtained in FIG. 1 .
- X-axis indicates the
- FIG. 3 related to Example 1 , shows in the Y-axis the concentration in micrograms/ml of CLNA obtained using as a bacterial medium an infant formula (Blemil-Plus-1 Forte®, LABORATORIOS ORDESA, S.L.).
- the number under the bars relate to the inoculated strains Lactobacillus oris ORD0255 (identified as 255) , Bifidobacterium breve ORD0123 (identified as 123), Bifidobacterium breve ORD0124 (identified as 124), Bifidobacterium breve ORD0128 (identified as 128), Bifidobacterium breve ORD134 (identified as 134), Bifidobacterium breve ORD0138 (identified as 138), Bifidobacterium breve ORD0294 (identified as 294), Bifidobacterium bifidum ORD0202 (identified as 202).
- strain used in dairy or agroalimentary industry any strain of a bacteria that is traditionally known as a “food-grade bacteria”, which means that can be used in the processes for obtaining food, or as active principles in any edible composition.
- strains used in dairy (milk) or agroalimentary industry there are the Bifidobacteria and the Lactic Acid Bacteria (LAB), including among others the genus Lactobacillus, and
- milk or dairy derivative or “milk-based compound” or “milk-based product”, used herewith interchangeable, is to be understood any composition or compound derived from milk of any source and origin, including animal or vegetal milk, such as milk (entire, skimmed, semi-skimmed) in liquid form or as a powder, a yogurt, a curd, a cheese, an infant formula, and an ice-cream.
- Said dairy product can be used as an ingredient of a composition containing other edible ingredients, as well as a source of carbon or other compounds, namely as a source of linolenic acid.
- the "fat portion” or "fat fraction” is to be understood any composition or compound derived from milk of any source and origin, including animal or vegetal milk, such as milk (entire, skimmed, semi-skimmed) in liquid form or as a powder, a yogurt, a curd, a cheese, an infant formula, and an ice-cream.
- Said dairy product can be used as an ingredient of a composition containing other
- milk fat is secreted in the form of a fat globule surrounded by a membrane.
- Each fat globule is composed almost entirely of triacylglycerols and is surrounded by a membrane consisting of complex lipids such as phospholipids, along with proteins. These act as emulsifiers which keep the individual globules from coalescing and protect the contents of these globules from various enzymes in the fluid portion of the milk.
- lipids are triacylglycerols
- di- and monoacylglycerols small amounts of di- and monoacylglycerols, free cholesterol and cholesterol esters, free fatty acids, and phospholipids are also present.
- fat composition in milk varies widely in the composition due to genetic, lactation, and nutritional factor difference between different species.
- the fat-soluble vitamins A, D, E, and K along with essential fatty acids such as linoleic acid and linolenic acid are also found within the milk fat portion, from animal or vegetal origin, of the milk.
- a "fat portion” or “fat fraction” (interchangeable herewith) contained in a milk or a milk-based product is to be understood as the fraction of lipidic components that could be from vegetal origin, which is for example, added in some infant formulas.
- enzyme activity profile is to be understood the pool of enzymes that are active in a cell system, in particular in a bacterial strain, and which give raise to a determined phenotype.
- probiotic is to be understood in the sense of the present invention as live microorganisms which when administered in adequate amounts confer a health benefit on the host.
- the known benefits of enteral administration of probiotic microorganisms include enhanced host defense to disease improving the properties of the indigenous microbiota and increasing colonization resistance to the harmful microbiota.
- Probiotics have been suggested to play an important role in the formation or establishment of a well-balanced, indigenous, intestinal microbiota in newborn children or adults receiving high doses of antibiotics. Lactic acid bacteria, especially specific strains of Lactobacillus and species of Streptococcus, Enterococcus and
- Bifidobacterium genera have been recommended for their use as probiotics.
- a "mutant of a bacterial strain or variant thereof encompasses bacteria obtained by mutation, variation or recombination of each of the strains, provided that the resulting bacteria have the activity of growing to an optical density of at least 0.9 (said optical density determined at 600 nm, OD 6 oo , and at 48 hours after initial of growing in anaerobiosis), in a Man Rogosa Sharpe broth medium, as well as in a medium comprising at least the fat portion, from animal or vegetal origin, contained in a milk-based product, both media comprising at least 0.3 mg/ml of linolenic acid in the medium; and being able to produce conjugated linolenic acid (CLNA) in both media.
- CLNA conjugated linolenic acid
- the mutant is a genetically modified mutant obtained by classical mutagenesis (i.e. using chemical or physical agents) or by genetic engineering techniques.
- the variant is a naturally occurring variant.
- pharmaceutically acceptable as used herein pertains to
- Suitable carriers, excipients, etc. can be found in standard pharmaceutical texts, and include, as a way of example preservatives, agglutinants, humectants, emollients, and antioxidants.
- terapéuticaally effective amount means an amount of an active agent (ingredient) high enough to deliver the desired benefit, either the treatment or prevention of the illness, but low enough to avoid serious side effects within the scope of medical judgment.
- anti-obesity agent any compound or product (including pharmaceutical active principles, and bacterial strains per se or as ingredients in pharmaceutical compositions), which prevents obesity or even allows treating obesity.
- immunomodulator agent relates to any compound, bacterial strain or composition that is able to promote or to regulate the balanced functioning of the immune system.
- balanced functioning is to be understood that the system does not lead to autoimmune diseases, as well as does not conduct to any immune-depressed situation.
- any ranges given include both the lower and the upper end-points of the range.
- the method of the invention allows obtaining high amounts of the isomers of CLNA.
- the bacillus strains of lactic acid bacteria and of bifidobacteria used in the method are further characterized by comprising the following enzymatic activity profile:
- (+) means that the enzymatic activity is present, and (-) means that the enzymatic activity is not present;
- API ZYM API system, BioMerieux
- the determination of the growing of the LAB and Bifidobacteria strains is performed by determining the Optical Density (OD) with a spectrophotometer at a wavelength of 600 nm of a growing culture comprising the desired medium (MRS or a medium with a milk-based product) and the strain
- determination of the optical density of the growing culture is done at 48 hours after initial of the culture growing.
- the linolenic acid comprised in the media is provided as a supplement of the media or it is a constituent of the elected media, in particular it can be used the linolenic acid provided in the at least the fat portion (from animal or vegetal origin) contained as component or ingredient in a milk-based product.
- the method is performed in a medium comprising a milk-based product selected from the group consisting of milk (entire, skimmed, semi-skimmed) in liquid form or as a powder, a yogurt, a curd, a cheese, an ice-cream, a milk infant formula, and mixtures thereof.
- a milk-based product selected from the group consisting of milk (entire, skimmed, semi-skimmed) in liquid form or as a powder, a yogurt, a curd, a cheese, an ice-cream, a milk infant formula, and mixtures thereof.
- the milk-based product is a reconstituted powder milk, in particular a reconstituted skim milk powder.
- the milk-based product is a milk infant formula.
- the milk-based product can be of any origin, such as milk from cow, goat, or sheep. Indeed, the milk-based product has to comprise at least a fat portion (from animal or vegetal origin).
- the conversion rate is comprised from 60 % to 99 % using any of the bacteria selected from LAB or bifidobacteria strains of the species Bifidobacterium breve, Bifidobacterium bifidum and Lactobacillus oris.
- the conversion rate is comprised from 30 % to 80 %.
- the conversion rate of LNA to CLNA in a milk infant formula is comprised from 70 % to 80 %.
- the conjugated linolenic acid obtained is the isomer C18:3 c/s-9 trans- 1 c/s-15.
- This isomer in a mixture with the isomer c/s-9 frans-13 c/s-15 has been reported as anti- obesity agent due to its capacity of increasing lipid mobilization in adipose tissue.
- the percentage by weight of the isomer C18:3 cis- 9 trans ⁇ 1 c/s-15 conjugated linolenic acid is comprised from 75 % (w/w) to 98 % (w/w) referred to the total amount of conjugated linolenic acid obtained in the method.
- the mass or amount (weight) of CLNA isomers are measured by gas chromatography after direct
- the invention refers also to a method for preparing an isomer of conjugated linolenic acid (CLNA) from linolenic acid (LNA) in a medium comprising at least the fat portion from animal or vegetal origin contained in a milk-based product, said fat portion containing linolenic acid, the method comprising adding to the medium at least one strain used in dairy or agroalimentary industry, selected from the group consisting of a lactic acid bacteria (LAB) and a Bifidobacteria, said strain characterized by: i) being able to grow to an optical density of at least 0.9 OD 6 oo, determined at 48 hours after initial of growth in anaerobiosis, in a Man Rogosa Sharpe broth medium, as well as in a medium comprising at least the fat portion, from animal or vegetal origin, contained in a milk- based product, both media comprising at least 0.3 mg/m
- CLNA conjugated linolenic acid
- LNA l
- the strains produce Vitamin B12 and Vitamin B9.
- Several media can be used for growing the bacterial strains for the determination of these vitamins, with the proviso that these media are vitamin depleted or with a known concentration for determining the amount of vitamins produced by the effect of the tested strains.
- the strain used in dairy or agroalimentary industry is a strain of Bifidobacterium with the ability to grow in both media MRS, and a media comprising at least the fat portion (from animal or vegetal origin) or fraction contained as a component of the milk-based product, both media also comprising free LNA, which is added as a
- the LAB or Bifidobacteria is able to grow in a medium with a concentration of LNA of at least 0.3 mg/ml (w/v) in respect of the total volume wherein the analysis of the growing of the bacteria is performed.
- a preferred amount is selected from the group consisting of 0.3 mg/ml, 0.4 mg/ml, 0.5 mg/ml, 0.6 mg/ml, 0.7 mg/ml and 0.8 mg/ml.
- the concentration of LNA is 0.5 mg/ml.
- the strain of Bifidobacterium is selected from Bifidobacterium breve and Bifidobacterium bifidum.
- agroalimentary industry is a LAB strain, in particular is a strain of
- Lactobacillus with the ability to grow in both media MRS and a media comprising a milk-based product, both media also comprising LNA in a concentration of at least 0.3 mg/ml (w/v) in respect of the total volume wherein the analysis of the growth of the bacteria is performed.
- a preferred amount is selected from the group consisting of 0.3 mg/ml, 0.4 mg/ml, 0.5 mg/ml, 0.6 mg/ml, 0.7 mg/ml and 0.8 mg/ml.
- the strain is of Lactobacillus oris.
- Another embodiment of the invention is a method as disclosed above, wherein the strain is selected from the group consisting of:
- CECT Coleccion Espahola de Cultivos Tipo
- CECT under the Depositor's reference ORD255, provisional and definitively identified as CECT 8240, or a mutant or variant thereof, wherein the mutants or variants of said strains are obtained using one of the deposited strains as starting material, and wherein the mutant strains retain the essential properties of the deposited strains, wherein said essential properties are: i) the ability to grow to an optical density of at least 0.9 (OD 6 oo),
- Rogosa Sharpe broth medium as well as in a medium comprising at least the fat portion, from animal or vegetal origin, contained in a milk- based product, both media comprising at least 0.3 mg/ml of linolenic acid in the medium; and ii) being able to produce conjugated linolenic acid (CLNA) in both media.
- CLNA conjugated linolenic acid
- All the above-mentioned bacterial strains are strains used in dairy or agroalimentary industry, including the lactic acid bacteria (LAB), such as Lactobacillus, and Bifidobacteria. All the strains used in the method have the advantage of being able to produce CLNA in high amounts in several media, including not only in vitro growth bacterial media, but also in products derived from milk or milk-based products. This implies the additional advantage of being usable in compositions or in edible products containing milk that are further consumed by animals, including human. In addition all the strains used in the method of the invention, in particular those strains of
- Bifidobacteria and Lactobacillus have a bacillus morphology or shape, that is, they are rod-shape bacteria.
- All these bacteria can be used in the method of the invention, in which the source of free LNA is from the milk-based product itself (fat portion or fraction from animal or vegetal origin), or can be externally provided from other source or environment.
- the source of free LNA is from the milk-based product itself (fat portion or fraction from animal or vegetal origin), or can be externally provided from other source or environment. Examples include linseed (flaxseed), soybean, rapeseed (canola), and walnuts.
- the strains can produce CLNA in several media, for example in the context of a milk composition in the gastrointestinal tract.
- strains of the invention are producers of highly pure isomers of CLNA, in particular of an isomer that has been associated to the reduction of obesity risk. See for reference Miranda et al., "c/s-9, trans ⁇ 1 , c/s-15 and c/s-9, frans-13, c/s-15 CLNA mixtures activates PPARa in HEK293 and reduces triacylglycerols in 3T3-L1 cells", Lipids -201 1 , vol. 46(1 1 ), pp.: 1006- 1012.
- the strains according to the invention behave the additional advantage that they are also producers of other conjugated fatty acid isomers, such as C18:2 c/s-9, frans-1 1 conjugated linoleic acid (Rumenic acid, CLA) and low amounts of C18:2 frans-10, c/s-12, with interesting properties.
- CLA has been mainly related with increased immune function, but also as an anti-cancer agent or agent helpful against cancer (colorectal cancer), and as anti-atherosclerosis agent useful in cardiovascular diseases, for treating high blood pressure, high cholesterol and triacylglycerides levels, osteoporosis, insulin resistance, inflammation, food-induced allergic reactions and as agent for modulating body composition (lowering body fat, preserving muscle tissue).
- the invention refers to a method for preparing an isomer of conjugated linolenic acid (CLNA), wherein the strains of LAB and bifidobacteria produce CLA from LA in both media; MRS and in a medium comprising at least the fat portion contained in a milk-based product.
- CLNA conjugated linolenic acid
- the conversion rate of LA to CLA in both media was equal or higher than 35 % calculated as indicated before.
- the conversion rate of LA in both media when a strain of B. breve is used is comprised from 35 % to 85 %.
- Vitamin B9 is reported as immune modulator agent or immune enhancing agent, meanwhile vitamin B12, among others properties, is also employed as anti-obesity agent.
- Vitamin B9 deficiency during pregnancy can produce pregnancy complications including neural tube defects, increased risk of congenital heart defects, increased risk of preterm delivery, infant low birth weight and fetal growth retardation, also B9 deficiency has been related with increasing homocysteine level in the blood, which may lead to spontaneous abortion and pregnancy complications, such as placental abruption and pre-eclampsia Vitamin B12 deficiency can produce atrophic gastritis, pernicious anaemia, thyrotoxicosis; hemorrhage, liver and kidney disease, supplementation with B12 is prescribed after surgical removal of the stomach intestine or gastric bypass. Special populations at risk of deficiencies for B9 or B12 are vegetarians, women during pregnancy, elderly people and babies.
- Said LNA may be present in the same milk-based product (such as in a milk infant formula), or can be added as an additional ingredient in another edible product that comprises an amount of the milk-based product and other components.
- the source of LNA is the milk-based product itself, also comprising the strains
- this milk-based product be in a dehydrated form in which strains are deactivated.
- this milk-based product be in a dehydrated form in which strains are deactivated.
- this milk-based product be in the form of a lyophilized powder. Once rehydrated, for example by re-suspension in a liquid matrix, the bacterial strains become active and they are able to transform the LNA to CLNA.
- the scope of the present invention also encompasses bacteria obtained by mutation, variation or recombination of the strains Lactobacillus oris
- Bifidobacterium breve ORD0123 provisional and definitive accession number CECT 8241
- Bifidobacterium breve ORD0124 provisional and definitive accession number CECT 8242
- Bifidobacterium breve ORD0128 provisional and definitive accession number CECT 8239
- Bifidobacterium breve ORD0123 provisional and definitive accession number CECT 8241
- Bifidobacterium breve ORD0124 provisional and definitive accession number CECT 8242
- Bifidobacterium breve ORD0128 provisional and definitive accession number CECT 8239
- Bifidobacterium breve ORD0123 provisional and definitive accession number CECT 8241
- Bifidobacterium breve ORD0124 provisional and definitive accession number CECT 8242
- Bifidobacterium breve ORD0128 provisional and definitive accession number CECT 8239
- the mutant is a genetically modified mutant obtained by classical mutagenesis (i.e. using chemical or physical agents) or by genetic engineering techniques.
- the variant is a naturally occurring variant.
- the general use of the strains of the invention is in the form of viable cells, although this is not the only way of supplying the strains.
- the strains are in the form of viable cells this means the introduction of the living bacteria in a milk-based product, or in an environment comprising a milk-based product. In this way, the administration of such products allows obtaining CLNA if in the media there is also a source of LNA.
- the present invention provides strains of lactic acid bacteria and bifidobacteria selected from the species of the group consisting of Bifidobacterium breve, Bifidobacterium bifidum, and Lactobacillus oris.
- the invention also relates to mixtures of any of the strains of the invention with another strain of the Bifidobacterium genus and/or Lactobacillus genus.
- the mixture comprises any of the strains of the invention and a strain of the species Bifidobacterium breve and/or Bifidobacterium longum and/or Bifidobacterium bifidum and/or
- the strains of the invention may be used for the preparation of a variety of food products, such as a milk products, a yogurt, a curd, a cheese (e.g. quark, cream, processed, soft and hard), a fermented milk, a milk powder, a milk based fermented product, an ice-cream, a fermented cereal based product, a milk based powder, a beverage, a dressing, and a pet food.
- food products e.g. liver paste, frankfurter and salami sausages or meat spreads
- chocolate spreads fillings (e.g.
- chocolate e.g. caramel, fondants or toffee
- baked goods cakes, pastries
- sauces and soups e.g., soups, fruit juices and coffee whiteners.
- food product is used herein in its broadest meaning, including any type of product, in any form of presentation, which can be ingested by an animal, including humans.
- the concentration of LNA in the food product is of at least 0.3 mg/ml or g of the edible (food) composition.
- the edible composition comprises 0.5 mg/ml or g of LNA.
- the desired amount of LNA may be provided by the accompanying ingredients in the matrix of the selected food (i.e., in a fat portion of a skimmed cow milked).
- the food does not contain LNA as raw ingredient, it can be added to the food composition as a supplement.
- the strains of the invention may be used for the preparation of a variety of nutritional compositions.
- Particular embodiments are a dietary supplement, an additive, and an infant formula.
- Dietary supplements intend to supply nutrients (vitamins, minerals, fatty acids or amino acids) that are missing or not consumed in sufficient quantity in a person's diet (infants, pregnant women, elderly people, etc).
- the strain of the invention is homogenized with other nutrients (vitamins, minerals, fatty acids or amino acids) that are missing or not consumed in sufficient quantity in a person's diet (infants, pregnant women, elderly people, etc).
- the strain of the invention is homogenized with other nutrients (vitamins, minerals, fatty acids or amino acids) that are missing or not consumed in sufficient quantity in a person's diet (infants, pregnant women, elderly people, etc).
- the strain of the invention is homogenized with other nutrients (vitamins, minerals, fatty acids or amino acids) that are missing or not consumed in sufficient quantity in a person's diet (in
- ingredients such as cereals or powdered milk to constitute an infant formula.
- the concentration of LNA is of at least 0.3 mg/ml or g of the nutritional composition.
- the edible composition comprises 0.5 mg/ml or g of LNA.
- the strain is present in an amount from about 10 5 cfu/g to about 10 9 cfu/g of the composition, and preferably in an amount of 10 7 cfu/g, according to the current legislation.
- the abbreviation "cfu” shall designate a "colony forming unit” that is defined as number of bacterial cells as revealed by microbiological counts on agar plates. Depending on the product, bacteria will be in viable form or non-viable.
- the present invention also provides pharmaceutical compositions comprising the strain of the invention, together with pharmaceutical excipients and/or carriers.
- the pharmaceutical composition may be prepared in form of tablets, dried oral supplements, dry tube feeding, etc., with the amount of bacteria to be incorporated therein being in the range of 10 7 cfu/g to about 10 11 cfu/g of product, and preferably in an amount of 10 9 cfu/g. Based upon the desired objective the person skilled in the art will select the appropriate excipients and/or carriers. Dried preparations are preferred because they have a better stability.
- the concentration of LNA is of at least 0.3 mg/ml or g of the pharmaceutical composition. In another embodiment, the pharmaceutical composition comprises 0.5 mg/ml or g of LNA.
- compositions of the invention may comprise the bacteria of the invention as single probiotic agent against obesity, combinations of such probiotics or combinations with other therapeutic/nutraceutical agents depending on the condition.
- the invention also provides a strain used in dairy or agroalimentary industry selected from Lactic acid bacteria (LAB) (in particular Lactobacillus) and Bifidobacteria, said strains selected from the species of the group consisting of, Bifidobacterium breve, Bifidobacterium bifidum, and
- LAB Lactic acid bacteria
- Bifidobacteria said strains selected from the species of the group consisting of, Bifidobacterium breve, Bifidobacterium bifidum, and
- Lactobacillus oris wherein the strain is characterized by: i) being able to grow to an optical density of at least 0.9 OD 6 oo,
- Example 1 Production of conjugated fatty acids with selected LAB strains. A screening was performed with 65 strains of LABORATORIOS ORDESA,
- the strains were able to provide great conversion rates independently of the tested media.
- other conjugated fatty acids were obtained with high yields.
- the strains were tested for its capacity of obtaining the conjugated fatty acids in reconstituted skimmed milk and in an infant formula (Blemil-Plus-1 Forte® of LABORATORIOS
- Table 1 .1 shows the optical density at 600 nm (Spectrophotometer Benchmark Plus, Biorad) measured at 37 °C of the mixtures of the strains in MRS supplemented with linoleic acid (LA, 0.5 mg/ml).
- Table 1 .1 Optical density at 48 H with LA in MRS.
- Table 1 .2 shows the optical density at 600 nm (Spectrophotometer Benchmark Plus, Biorad) measured at 37 °C of the mixtures of the strains in MRS supplemented with linolenic acid (LNA, 0.5 mg/ml).
- next table 1 .3 shows the optical density at 24 H at 600 nm
- Methodology for obtaining CLNA Bacteria for the screening and other assays realized a posteriori were activated in 10 ml of MRS (Pronadisa, Madrid, Espaha) supplemented with 0.25 % (weight/volume; w/v) of L-cysteine (Sigma) and 0.2% (w/v) of Tween- 80 (Scharlau, Sentmenat, Barcelona, Espaha). The strains were incubated over-night at 37 °C in anaerobiosis (Anaerobiosis chamber, Bactronll from Shellab, Cornelius, Oregon, USA).
- strains were reinoculated at 2.5 % at the same conditions with a supplement of LA or LNA at a final concentration of 0.5 mg/ml.
- the cultures were incubated for 24 hours at 37 °C in anaerobic conditions.
- the content of the methyl esters was analyzed by gas chromatography after derivatization of 500 ⁇ of the culture medium by direct transmethylation.
- Bifidobacterium breve 26M2 strain As positive control a Bifidobacterium breve 26M2 strain was used, known to be able to produce CLA and CLNA in MRS. All the assays were performed in triplicate.
- the strain Bifidobacterium breve 26M2 is the one identified in Jimenez E, et al., "Complete genome sequence of Bifidobacterium breve CECT 7263, a strain isolated from human milk", J Bacteriol. 2012,
- Table 2 shows the list of strains selected for being able to transform LNA (and LA) to CLNA (and CLA) in the three different tested media. In this table it is also indicated other features of the strains or other compounds obtainable with the method of the invention being performed with bacterial strains. The concentration of CLA and CLNA obtained in each media is also included.
- Vitamins B12 and B9 (folic acid) in the selected strains were measured using a commercial system, VitaFast Vitamin B12
- FIG. 1 shows the percentage of conversion of LNA to CLNA when the method of the invention is performed in the presence of each strain and using a medium comprising RSM as the milk-based product. Numbers over the bars indicate the value of the calculated conversion rate.
- conversion rates were all near 100 % in the strains of the species Bifidobacterium breve, Bifidobacterium breve and Lactobacillus oris. That is, a conversion rate comprised from 30 % to 100 %. In particular from 60 % to 99 %.
- FIG. 2 shows the percentage by weight of the isomer C18:3 c/s-9 trans- 1 c/s-15 in relation with the total weight of CLNA obtained and indicated in Table 2.
- FIG. 3 shows the concentration of CLNA (pg/ml) obtained as a mean of the three assays performed with each strain. Numbers over the bars indicate the concentration obtained for each strain (and correspond with the ones in Table 2).
- the obtained concentrations were comprised from 81 .1 to 201 .3 g/ml.
- the conversion rates (not shown) were comprised from 30 % to 80 %.
- all the strains showed a conversion rate of 75 %, except of the methods performed with strains Lactobacillus oris ORD0255 (CECT 8240), and Bifidobacterium bifidum OR0D202 (CECT 8245), in which a conversion rate of 33.7 % and 36.9 % were obtained.
- Example 2 Isolation and characterization of lactic acid bacteria (LAB) (Lactobacillus) and Bifidobacteria. 2.1 . Isolation of microorganisms
- Bifidobacterium bifidum ORD0202 provisional and definitive accession number CECT 8245
- Bifidobacterium breve ORD0123 provisional and definitive accession number CECT 8241
- Bifidobacterium breve ORD0124 provisional and definitive accession number CECT 8242
- Bifidobacterium breve ORD0128 provisional and definitive accession number CECT 8239
- Bifidobacterium breve ORD0134 provisional and definitive accession number CECT 8243
- Bifidobacterium breve ORD0138 provisional and definitive accession number CECT 8244
- Bifidobacterium breve ORD0294 provisional and definitive accession number CECT 8246
- have been isolated from babies feces and Lactobacillus oris ORD0255 provisional and definitive accession number CECT 8240
- DNA templates were amplified by the polymerase chain reaction (PCR) on a thermocycler, using universal primers amplifying a 348 bp region of the 16S rRNA gene, Y1 : 5 ' -TGG CTC AGG ACG AAC GCT GGC GGC-3 ' (SEQ ID NO: 0
- 16s1 a 5 ' -AAT ACA TGC AAG TCG AAC GA-3 ' (SEQ ID NO: 1 1 )
- 16s1 b 5 ' -TTA ACC CAA CAT CTC ACG AC-3 ' (SEQ ID NO: 12).
- the amplification mixture (50 ⁇ ) comprised 1 .5 ⁇ (100 pmol/ ⁇ ) ofeach of Y1 and Y2 primers or 16s1 a and 16s1 b, 1 ⁇ (1 U/ ⁇ ) of Taq DNA Polymerase KOD Hot Start (Merck), 5 ⁇ of 10* KOD Hot Start buffer (Merck), 5 ⁇ of dNTP mixture (containing 1 mM each of dATP, dGTP, dCTP and dTTP, Merck), 3 ⁇ of 25mM MgSO4 (Merck), 31 ⁇ of sterile filtered water (Milli-Q purification system, Millipore) and 2 ⁇ of DNA template.
- the DNA templates were amplified using Y1 and Y2 primers by initial denaturation at 95° C for 3 min, followed by 30 cycles of denaturation at 95° C for 2.3 min, annealing at 70° C for 30 sec, extension at 70° C for 40 sec, and a final extension at 70° C for 7 min.
- the DNA templates were amplified using 16s1 a and 16s1 b oligos by initial denaturation at 95° C for 2 min, followed by 30 cycles of denaturation at 95° C for 1 min, annealing at 61° C for 30 sec, extension at 72° C for 1 .30 min, and a final extension at 72° C for 10 min. Controls, devoid of DNA, were simultaneously included in the amplification process.
- the integrity of PCR products was assayed by the development of single bands following electrophoresis for 1 h at 100 V in 2% or 1 % (w/v) agarose gels in tris-borate EDTA buffer.
- Amplicons were purified using a commercial kit and subsequent sequencing reactions were performed using the Big Dye Terminator v3.1 cycle
- Sequencing primers were the same ones used in the amplification reaction but diluted ten times (5 pmol). The resulting sequences were automatically aligned for each strain and then inspected by eye. The resulting 16S rRNA gene sequences were compared in a BLAST search with those in the National Library of Medicine database.
- SEQ ID NO:1 to SEQ ID NO:8 correspond to the 16S rRNA of any of the strains, amplified with primers of sequences SEQ ID NO: 1 1 and 12, and they correlate with the identification of the strains as follows:
- SEQ ID NO:1 correlates with Bifidobacterium breve ORD0123 (provisional and definitive accession number CECT 8241 ):
- SEQ ID NO:2 correlates with Bifidobacterium breve ORD0124 (provisional and definitive accession number CECT 8242):
- TACCCTGGTAGTCCACGCCGTA SEQ ID NO:3 correlates with Bifidobacterium breve ORD0128 (provisional and definitive accession number CECT 8239):
- SEQ ID NO:5 correlates with Bifidobacterium breve ORD0138 (provisional and definitive accession number CECT 8244): TTGCGGCATGGGATGGGGTCGCGTCCTATCAGCTTGATGGCGGGGTAA CGGCCCACCATGGCTTCGACGGGTAGCCGGCCTGAGAGGGCGACCGG CCACATTGGGACTGAGATACGGCCCAGACTCCTACGGGAGGCAGCAGT GGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCGACGCCGCGTG AGGGATGGAGGCCTTCGGGTTGTAAACCTCTTTTGTTAGGGAGCAAGGC ATTTTGTTGAGTGTACCTTTCGAATAAGCACCGGCTAACTACGTGCCA GCAGCCGCGGTAATACGTAGGGTGCAAGCGTTATCCGGAATTATTGGGC GTAAAGGGCTCGTAGGCGGTTCGTCGCGTCCGGTGTGAAAGTCCATCG CTTAACGGTGGATCCGCGCCGGGTACGGGCGGGCTTGAGTGCGG
- SEQ ID NO:6 correlates with Bifidobacterium bifidum ORD0202 (provisional and definitive accession number CECT 8245): TCCTGGAAACGGGTGGTAATGCCGGATGTTCCACATGATCGCATGTGAT
- ATTTATTGGGCGTAAAGGGCTCGTAGGCGGCTCGTC SEQ ID NO:7 correlates with Lactobacillus oris ORD0255 (provisional CECT and definitive accession number 8240): G G G G ATAACATTTG G AAACAG GTGCTAATACCGC ATAACTTG G AAAACCA CATG GTTTTCCAATAAAAG ATG GTTTCG GCTATCACTTTG G G ATG G GCCC GCGGTGCATTAGCTAGTTGGTAAGGTAACGGCTTACCAAGGCGATGATG CATAGCCGAGTTGAGAGACTGATCGGCCACAATGGAACTGAGACACGGT CCATACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGGCGCA AGCCTGATGGAGCAACACCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAA AACTCTGTTGTTGGAGAAGAACGTGCGTAAGAGTAACTGTTTACGCAGTG ACGGTATCCAACCAGAAAGTCACGGCTAACTACGTGCCAGCAGCCGCGG
- SEQ ID NO:8 correlates with Bifidobacterium breve ORD0294 (provisional and definitive accession number CECT 8246):
- CGC Caenorhabditis Genetics Centre
- NGM nematode growth medium
- Escherichia coli OP50 strain was used as normal nematode diet and was also provided by the CGC. Worms were grown on NGM using E. coli OP50 as control diet
- NGM plates as control media
- NGM plates with 6 g/mL of Orlistat Sigma-Aldrich, Madrid, Spain
- Nile Red 9-diethylamino-5H-benzo[a]phenoxazin-5-one, Sigma, St. Louis, MO, USA
- the dye was added on the top of the NGM agar plates, preseeded with
- Escherichia coli OP50 to a final concentration of 0.05 g/mL. Worms were incubated at 20 °C for 3 days until young adult stage. After this incubation period, nematode samples were placed in M9 buffer and fluorescence was measured in an FP-6200 system (JASCO Analytical Instruments, Easton, MD, USA) using ⁇ excitation 480 nm and ⁇ emission 571 nm.
- strains can be supplied in an edible product (infant formula in particular) aimed to control or prevent obesity development in children and babies.
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EP13817928.8A EP2935600A2 (en) | 2012-12-21 | 2013-12-20 | Process for producing conjugated linolenic acid from linolenic acid employing bifidobacterium breve, bifidobacterium bifidum, or lactobacillus oris strains |
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PCT/EP2013/077671 WO2014096352A2 (en) | 2012-12-21 | 2013-12-20 | A microbiological method for obtaining conjugated linolenic acid from linolenic acid, and bacterial strains for performing the method |
EP13817928.8A EP2935600A2 (en) | 2012-12-21 | 2013-12-20 | Process for producing conjugated linolenic acid from linolenic acid employing bifidobacterium breve, bifidobacterium bifidum, or lactobacillus oris strains |
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EP13817928.8A Withdrawn EP2935600A2 (en) | 2012-12-21 | 2013-12-20 | Process for producing conjugated linolenic acid from linolenic acid employing bifidobacterium breve, bifidobacterium bifidum, or lactobacillus oris strains |
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Non-Patent Citations (2)
Title |
---|
GORISSEN, L. ET AL.: "Production of conjugated linoleic acid and conjugated linolenic acid isomers by Bifidobacterium species", APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, vol. 87, no. 6, 17 June 2010 (2010-06-17), pages 2257 - 2266, XP019841711 * |
HENNESSY, A.A. ET AL.: "The Production of Conjugated alpha-Linolenic, gamma-Linolenic and Stearidonic Acids by Strains of Bifidobacteria and Propionibacteria", LIPIDS, vol. 47, no. 3, 10 December 2011 (2011-12-10), pages 313 - 327, XP035017377, DOI: 10.1007/S11745-011-3636-Z * |
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