ES2277546A1 - Microbial method for the production of specific isomers of conjugated linoleic acids - Google Patents

Abstract

The invention relates to a microbial method for the production of specific isomers of conjugated linoleic acids, particularly the cis-9, trans-11 isomer, using micro-organisms. The inventive method involves the optional incorporation of the trans-11 isomer of vaccenic acid as a substrate for a culture medium in which micro-organisms such as bacteria, yeast and fungi generate the bioconversion to the isomer of interest with the generation of a novel Z (cis) configuration at position 9 of the chain. The aforementioned conjugated linoleic acids can be used in the bio-food industry and the biotechnology sector.

Description

Microbial production process of specific isomers of conjugated linoleic acids.

Technical sector

This invention relates to a method of production of conjugated linoleic acids, particularly the cis-9 isomer, trans-11, using microorganisms The use of these acids can be applied to the bio-food and biotechnology sector.

State of the art

With the name of conjugated linoleic acids (abbreviated CLAs for the acronym in English of c onjugated l inoleic a cids), a whole collection of positional and geometric isomers of polyunsaturated fatty acids (abbreviated PUFAs) is usually referred to as p oly u nsaturated f atty a cids) with chain length of 18 carbon atoms whose double bonds, unlike the rest of PUFAs, are conjugated in the molecule by occupying consecutive positions.

The name that fatty acids have with 18 carbon atoms and two double bonds carbon = carbon is acidic octadecadienoic. Of these molecules there are numerous isomers, both position of the double bonds, called "isomers of position ", as of stereochemistry (" isomers geometric, "cis or trans, Z or E). Among them there are two of relevant importance It is the so-called cis-9, trans-11- and trans-10, cis-12-.

The chemical differences between linoleic acid and these two isomers imply drastic changes in their biological properties. In the case of CLAs, relevant functional activities are attributed that may be of radical interest to the agri-food industry involved in the production of additives and functional ingredients. For example, CLAs have been shown to be able to act as preventive and therapeutic agents in many tumor models in experimental animals (Margot M et al . (2003). J. Mammary Gland Biol. Neoplas. 8 : 103-118) . Specifically, CLA inhibition of the induction of breast carcinogenesis has been demonstrated (Ip C et al . (1991). Cancer Res. 51 : 6118-6124; Ip et al . (1997). Carcinogenesis 18 : 755-759; Thompson H et al . (1997) Cancer Res. 57 : 5067-5072; Ip C et al . Cancer Res. 54 : 1212-1215; and Ip C et al . Nutr. Cancer 24 : 149-157) and colon cancer in rat models (Ha YL et al . (1990). Cancer Res. 50 : 1097-1101; Park HS et al . (2001). Br. J. Nutr. 86 : 549-555; and Liew C et al . ( 1995). Carcinogenesis 16 : 3037-3043). In addition, an effect of CLAs on the reduction of size and metastasis in animal models of prostate cancer, lung cancer and breast cancer has been observed. Using in vitro culture models of human cancer cells, tumor proliferation inhibition has been observed using the cis-9-trans-11 isomer. This isomer added in diet is capable of reducing the occurrence of breast carcinogenesis in rats (Cori BA et al . (2003). J Nutr. 133 : 2893-2900). In addition, during the last months the molecular mechanisms responsible for all these anticancer effects have begun to be unraveled (Chujo H et al . (2003). Cancer Lett. 202 : 81-87; Kim KH et al . (2003). Nutrition 19 : 772-777; Miller et al . (2003). Br. J. Nutr. 90 : 877-885; Kemp MQ et al . (2003). J. Nutr. 133 : 3670-3677; Chen BQ et al . ( 2003). World J Gastroenterol. 9 : 1909-1914; Park HS et al . (2004). J. Nutr. Biochem. 15 : 229-235; Tanmahasamut P et al . (2004). J. Nutr. 134: 674 -680; and Nichenametla S et al . (2004). J. Toxicol. Environ. Health A 67 : 469-481).

To all this we must add other well-known biological activities of CLAs. The most commercially exploited refers to the fact that dietary intake reduces fat stores in rats (Park Y et al . (1997). Lipids 32 : 853-858; Delany JP et al . (1999 ) Am. J. Physiol. Regul. Integr. Comp. Physiol. 276 : R1172-R1179; Tsuboyama N et al . (2000). Diabetes 49 : 1534-1542; and Ohnuki K et al . (2001). Biosci. Biotechnol. Biochem. 65 : 2200-2204.), Mice (Stang) GI. (2000). J. Nutr. 130 : 1140-1146; Azain MJ et al . (2000). J. Nutr. 130 : 1548-1554; Sugano M et al . (2001). Biosci Biotechnol Biochem 65 : 2535-2541; Poulos SP et al . (2001). J. Nutr. 131 : 2722-2731; Sisk MB et al . (2001). J. Nutr. 131 : 1668-1674; and Ealey KN et al . (2002). Lipids 37 : 853-861), hamsters (from Deckere EA et al . (1999). Br. J. Nutr. 82 : 309-317) and pigs (Ostrowska E et al . (1999). J. Nutr. 129 : 2037-2042; and Thiel-Cooper RL et al . (2001). J. Anim. Sci. 79 : 1821-1828). Therefore, it is used in the formulation of some nutritional preparations with the idea of converting fat into muscle (diet for athletes) or losing weight without losing muscle (weight loss diets).

Other properties of CLAs refer to their ability to inhibit atherogenesis in hamsters and rabbits (from Deckere EA et al . (1999). Br. J. Nutr. 82 : 309-317; Lee KN et al . (1994). Atherosclerosis 108 : 19-25; Kritchevsky D et al . (2000). J. Am. Coll. Nutr. 19 : 472S-477S; and Wilson TA et al . (2000). Nutr. Res. 20 : 1795-1805, 32-34) and also, in pre-diabetic rats, their ability to increase insulin sensitivity and improve glucose tolerance (Houseknecht KL et al . (1998). Biochem. Biophys. Res. Commun. 244 : 678-682 ; and Ryder JW et al . (2001). Diabetes 50 : 1149-1157). However, this effect is attributable to the t-10-c-12 isomer that induces hepatic steatosis, which discourages its use (Clément L et al . (2002). J. Lipid Res. 43 : 1400-1409).

The natural biological source of linoleic acid They are vegetable oils. On the contrary, the biological source CLAs' natural products are dairy and meat from ruminants Actually in the latter case it is a bacterium of rumen who produces the CLAs and fixes them on the animal's tissue. Be markets a combined CLAs for the food industry (an isomolar mixture of the two isomers with 80% purity) and pure isomers are also sold although their price is very high, probably due to the difficulty of purification of both isomers

It is possible to chemically synthesize each of the two isomers but the process is complicated, expensive and ineffective, so that only 50% yield is obtained with contamination of the two isomers. There are descriptions in the literature that demonstrate the possibility of using the rumen bacterium Butyvibrio fibrisolvens , or the enzyme linoleoate isomerase purified from it, to carry out an enzymatic conversion of a precursor fatty acid into CLAs. There are also examples of the use of species of the genus Lactobacillus or the use of lipases from Candida cylindracea or Mucor miehei in the selective obtaining of CLAs (Warvel, S; Borgdorf, R; Biotechnology Letters, 2000, 22 : 1151-55), but Again, all these processes are ineffective.

A way of specifically producing the cis-9-trans-11 isomer, based on growing the genetically modified Saccharomyces cerevisiae yeast (EUROSCARF) in the presence of vaccenic acid (Spanish patent ES2204328-B) so that said monosaturated fatty acid is converted, has been described. specifically in the cis-9-trans-l1- isomer thanks to the action of a Δ9-desaturase encoded in the genome of this microorganism. Moreover, it has been described that in humans the intake of the trans-11- isomer of vaccenic acid results in its bioconversion in the cis-9-trans-11- isomer (Turpeinen et al . (2002). Am. J. Clin Nutr. 76 : 504-510).

Therefore, the technical problem that arises is the specific production of the cis-9, trans-11- isomer, effectively, given its proven biological utility and safety. The present invention describes the tests of five strains of bacteria, seven strains of yeasts Saccharomyces cerevisiae , seven strains of yeasts other than Saccharomyces and two species of fungi, to evaluate their production capacity of the cis-9 isomer, trans-11 of conjugated linoleic acid, both in the presence and absence of vaccenic acid. The results are a new procedure for obtaining cis-9, trans-11 acid from conjugated linoleic acid.

Description of the invention

The present invention describes several procedures for the specific production of the isomer cis-9-trans-11 of conjugated linoleic acid. The first one describes the incorporation of the trans-11 acid isomer vaccenic as a substrate for culture media where a bacteria, a yeast or a filamentous fungus and bioconversion microbial of said substrate in the isomer of interest measured by the generation of a new configuration link Z (cis) in the position 9 of the chain. The second is about production endogenous by some microorganisms of the isomer cis9-trans11 without precursors in the culture medium. The constitutive production of the trans-11 isomer of vaccenic acid by some yeasts

Detailed description of the invention

The present invention is based on several facts related to the microbial metabolism of fatty acids. So, the addition as a substrate of the trans-11 isomer of vaccenic acid to different microorganisms allows the production of a polyunsaturated fatty acid (the isomer cis-9-trans-11 of conjugated linoleic acid) with additional unsaturation of configuration Z (cis) at position 9 of the chain of said substrate caused by the performance of the enzyme Δ 9 -desaturase present in all microorganisms tested. Even more, certain microorganisms they are capable of producing said isomer without substrate any precursor to contain fatty acids (vaccenic acid, acid oleic or others) in your cytoplasm and / or cell membranes that may act as substrates of enzyme activities above mentioned. In this sense, the production has been checked constitutive of the trans-11 acid isomer vaccenic by some microorganisms.

Detailed presentation of the realization

Without presupposing a limitation to the scope of the application of the present invention as defined in the present claims, examples are described below details of how to carry out the invention.

Example one

Isomer Production Procedure cis-9-trans-11 of linoleic acid conjugated with different microorganisms by incorporation of the trans-11 acid isomer vaccenic as a substrate a) Cultures of the different microorganisms with the trans-11 isomer of vaccenic acid

In the present invention the microorganisms detailed in table 1.

Pre-growth for the generation of isolated colonies of bacteria of the Lactobacillus pentosus and Leuconostoc citreum species was performed on MRS-Agar solid medium plates inoculating a stretch mark and incubating at 30 ° C for 48 hours. Colonies of the species Bacillus subtilis and Escherichia coli were pre-grown on MC-Agar solid medium plates by incubating at 30 ° C and 37 ° C respectively, for 24 hours. Colonies of the species Zymomonas mobilis were grown in YPD-Agar solid medium by incubating at 30 ° C for 48 hours. The pre-growth of the yeasts of the Saccharomyces genus, as well as of the non- Saccharomyces genera, was performed on YPD-agar medium plates inoculating a stretch mark of the microbial suspension and incubating at 30 ° C for 48 hours. Similarly, the pre-growth of fungal colonies of the Aspergillus niger and Penicillium roqueforti species was carried out on PDA solid medium plates incubating at 30 ° C and 19 ° C respectively, for 6 days.

To prepare the induction medium, 5 µl of a 0.5 M ethanol solution of the trans-11 isomer of the vaccenic acid was added to 5 ml of liquid medium, using the same means used to obtain colonies or mycelium but without agar and with the addition of 1% v / v tergitol. Thus, the trans-11 isomer of vaccenic acid was at a final concentration of 0.5 mM. The mixture was inoculated with a colony isolated from the microorganism in question and incubated for 48 hours at the corresponding temperature in each case and with a stirring of 200 rpm, with the exception of the Leuconostoc citreum strain that was incubated without stirring.

b) Isomer extraction procedure cis-9-trans-11 of conjugated linoleic acid

The culture was transferred to a 10 ml tube and centrifuged at 4000 rpm for 5 minutes. The supernatant was removed and the cells were resuspended in 1.5 ml of distilled water and the suspension was transferred to a 2 ml eppendorf vial. Be centrifuged the culture under the same conditions, repeating another Once the washing operation with the same volume of distilled water. It was finally centrifuged at 5000 rpm for 5 minutes to remove totally water. To the cells obtained 1 ml of CHCl3: MeOH (2: 1) and the mixture was allowed to incubate for 1 hour. Finally, it was centrifuged at 13200 rpm for 15 seconds and the organic extract was transferred to a new 2 ml vial where evaporated the solvent.

The fatty acid residue was methylmed by adding 500 µL of a 0.5 N solution of KOH in MeOH, followed by neutralization after 30 minutes with 500 µl of 1 N HCI and extraction with 500 µl hexane. The organic layer is separated and concentrated to an approximate volume of 20 µl.

The isomer was then analyzed cis-9-trans-11 of conjugated linoleic acid. The methyl ester was analyzed by gas chromatography-mass spectrometry (CG-EM) on an HP-VOC apolar column  (0.2 mm * 30 m * 1.12 \ mum) with the following program of temperatures: 80 ° C (0 min.); 2.5 ° C / min up to 220 ° C (0 min.); 2.0 ° C / min up to 260 ° C (15 min.).

Results

As indicated in Table 2, in all microorganisms the cis-9-trans-11- isomer of conjugated linoleic acid was produced to a greater or lesser extent. In the case of B. subtilis and L. pentosus bacteria, similar productions to those obtained in some yeasts of the Saccharomyces genus were detected, while in the rest of bacterial species the production was much lower. In the case of filamentous fungi, the production of the cis-9-trans-11- isomer was low. On the contrary, in all the non- Saccharomyces levaduriform species analyzed, except in the case of T. delbrueckii , high levels of production were detected, particularly in the case of P. anomala, P. jadinii and H. guillermondii species.

Example 2

Isomer Production Procedure cis-9-trans-11 of conjugated linoleic acid with different microorganisms when growing in culture media without fatty acid supplementation a) Cultures of the different microorganisms without the trans-11 isomer of vaccenic acid

As in the previous example, the microorganisms detailed in table 1.

Pre-growth for the generation of isolated colonies of bacteria of the Lactobacillus pentosus and Leuconostoc citreum species was performed on MRS-Agar solid medium plates inoculating a stretch mark and incubating at 30 ° C for 48 hours. Colonies of the species Bacillus subtilis and Escherichia coli were pre-grown on MC-Agar solid medium plates by incubating at 30 ° C and 37 ° C respectively, for 24 hours. Colonies of the species Zymomonas mobilis were grown in YPD-Agar solid medium by incubating at 30 ° C for 48 hours. The pre-growth of the yeasts of the Saccharomyces genus, as well as of the non- Saccharomyces genera, was performed on YPD-agar medium plates inoculating a stretch mark of the microbial suspension and incubating at 30 ° C for 48 hours. Similarly, the pre-growth of fungal colonies of the Aspergillus niger and Penicillium roqueforti species was carried out on PDA solid medium plates incubating at 30 ° C and 19 ° C respectively, for 6 days.

A colony isolated from the microorganism in question was inoculated in 5 ml of liquid medium with 1% tergitol (v / v) and the mixture was incubated for 48 hours at the corresponding temperature for each microorganism and with a stirring of 200 rpm, except of the Leuconostoc citreum strain that was incubated without agitation.

b) Extraction procedure of the possible isomer cis-9-trans-11 of conjugated linoleic acid produced

The culture was transferred to a 10 ml tube and centrifuged at 4000 rpm for 5 minutes. The supernatant was removed and the cells were resuspended in 1.5 ml of distilled water and the suspension was transferred to a 2 ml eppendorf vial. Be centrifuged the culture under the same conditions, repeating another Once the washing operation with the same volume of distilled water. It was finally centrifuged at 5000 rpm for 5 minutes to remove totally water. To the cells obtained 1 ml of CHCl3: MeOH (2: 1) and the mixture was allowed to incubate for 1 hour. Finally, it was centrifuged at 13200 rpm for 15 seconds and the organic extract was transferred to a new 2 ml vial where evaporated the solvent.

The fatty acid residue was methylmed by adding 500 µL of a 0.5 N solution of KOH in MeOH, followed by neutralization after 30 minutes with 500 µl of 1 N HCI and extraction with 500 µl hexane. The organic layer is separated and concentrated to an approximate volume of 20 µl.

The isomer was then analyzed cis-9-trans-11 of conjugated linoleic acid. The methyl ester was analyzed by gas chromatography-mass spectrometry (CG-EM) on an HP-VOC apolar column  (0.2 mm * 30 m * 1.12 \ mum) with the following program of temperatures: 80 ° C (0 min.); 2.5 ° C / min up to 220 ° C (0 min.); 2.0 ° C / min up to 260 ° C (15 min.).

Results

Given the high productions of the cis-9-trans-11 isomer detected in some of the microorganisms tested in the previous example, especially P. anomala and P. jadinii that produce more isomer (0.9748 mM and 0.6077 mM, respectively) that the amount of trans-11 precursor substrate of the added vaccenic acid (0.5 mM), it was decided to test the possible endogenous de novo synthesis by the microorganisms analyzed. For this, they were grown under the same conditions as in the previous example but without the trans-11 isomer of the vaccenic acid in the medium formulation.

The results obtained are shown in table 3 and indicate that only H. guillermondii and the species of the genus Pichia analyzed in the experiment produce the cis-9-trans-11 isomer at levels of relevance, especially in the case of P. anomala and P. jadinii . The rest of the strains tested have residual productions of the isomer in question.

Example 3

Production procedure of the trans-11 isomer of vaccenic acid when growing levaduriform species in culture a) Cultures of the different microorganisms

Strains S. cerevisiae CECT 1176 and Pichia anomala CECT 10590 (table 1) were used.

Yeast pre-growth was performed in YPD-agar media plates inoculating a stretch mark the microbial suspension and incubating at 30 ° C for 48 hours. A colony isolated from the microorganism in question was inoculated in 5 ml of YPD liquid medium with 1% tergitol (v / v). The mixture was incubated. for 48 hours at the corresponding temperature for each microorganism with a stirring of 200 rpm.

b) Extraction procedure of the possible isomer trans-11 of vaccenic acid

The culture was transferred to a 10 ml tube and centrifuged at 4000 rpm for 5 minutes. The supernatant was removed and the cells were resuspended in 1.5 ml of distilled water and the suspension was transferred to a 2 ml eppendorf vial. Be centrifuged the culture under the same conditions, repeating another Once the washing operation with the same volume of distilled water. It was finally centrifuged at 5000 rpm for 5 minutes to remove totally water. To the cells obtained 1 ml of CHCl3: MeOH (2: 1) and the mixture was allowed to incubate for 1 hour. Finally, it was centrifuged at 13200 rpm for 15 seconds and the organic extract was transferred to a new 2 ml vial where evaporated the solvent.

The fatty acid residue was methylmed by adding 500 µL of a 0.5 N solution of KOH in MeOH, followed by neutralization after 30 minutes with 500 µl of 1 N HCl and extraction with 500 µl hexane. The organic layer is separated and concentrated to an approximate volume of 20 µl.

The isomer was then analyzed trans-11 of vaccenic acid. The methyl ester is analyzed by gas chromatography-spectrometry of masses (GC-MS) in a nonpolar column HP-VOC (0.2 mm * 30 m * 1.12 \ mum) with the following temperature program: 80 ° C (0 min.); 2.5 ° C / min up to 220 ° C (0 min.); 2.0 ° C / min up to 260 ° C (15 min.).

Results

Table 4 shows the results obtained. As can be seen, yeast S. cerevisiae CECT 1176 produces a residual amount of the trans-11 isomeric isomer of vaccenic acid. In contrast, P. anomala CECT 10590 yeast produces approximately 0.5 mM of said compound, defining culture conditions for the production of this compound with possible activity as a functional ingredient. In any case, these results suggest that the production of the 9-cis, 11-trans- isomer of linoleic acids conjugated by the microorganisms of Table 1 is due to the fact that said microorganisms are, to a greater or lesser extent, producers of 11-trans- acid. vaccenic

TABLE 1 Microorganisms tested in the conversion of trans-11 isomer of vaccenic acid in the isomer cis-9-trans-11 of conjugated linoleic acid

100

TABLE 2 Production values of the isomer cis-9-trans-11 of conjugated linoleic acid obtained by different microorganisms growing in the presence of 0.5 mM of the isomer trans-11 of vaccenic acid. The results in mM represent the average of three experiments independent

101

TABLE 3 Production values of the isomer cis-9-trans-11 of conjugated linoleic acid obtained by different microorganisms growing without the addition of the isomer trans-11 of vaccenic acid. The results in mM represent the average of three experiments independent

102

TABLE 4 Production values of the trans-11 isomer of vaccenic acid obtained by S. cerevisiae CECT 1176 and P. anomala CECT 10590. The results in mM represent the average of three independent experiments

103

Isomer Use cis-9-trans-11 of Conjugated linoleic acid in the manufacture of yeast biomass baker and microflora yogurt

In preliminary experiments, a process for the production of baker's yeast biomass loaded with the cis-9-trans-11 isomer of conjugated linoleic acid has been carried out. Saccharomyces cerevisiae CECT 1326 yeast has been grown in a 300-liter fermenter containing 200 liters of rich medium with molasses as a source of carbon and the trans-11 isomer of vaccenic acid at a concentration of 0.5 mM. The fermentation conditions were a temperature of 30 ° C with stirring for 24 hours. After this time, the yeast-rich biomass enriched in the cis-9-trans-11 isomer was collected by centrifugation, which was washed twice by resuspension in 50 mM sodium phosphate buffer pH 6.8 and subsequent centrifugation. The resulting biomass pulp was characterized in terms of its content in the cis-9-trans-11 isomer of conjugated linoleic acid, determining a concentration of 0.16 mM. With these yeasts, bread was manufactured in a pilot oven following an established industrial protocol. The concentration of the cis-9-trans-11 isomer of the linoleic acid was evaluated on the breads produced, which was 0.35 µg / gram of bread.

Following a similar methodology has been carried carried out a procedure for the production of yogurt enriched in conjugated linoleic acids, characterized in that it consists of add to the growth medium of lactic acid bacteria corresponding the trans-11 isomer of the acid vaccenic Industrial productions have also been carried out of sausages cured using fermentation initiators (lactic acid bacteria) loaded with the isomer cis-9-trans-11 of conjugated linoleic acid by pre-growth in media with the trans-11 isomer of vaccenic acid.

Claims (7)

1. Procedure for the production of conjugated linoleic acids characterized by the following stages:

to)

incubation of a microorganism producing conjugated linoleic acids, to be chosen from the group consisting of bacteria, yeasts and fungi, preferably non- Saccharomyces yeasts, in liquid medium; Y

b)

linoleic acid extraction conjugates resulting from the incubation of step a); purification and characterization of said acids.

2. Method of production of conjugated linoleic acids according to claim 1, characterized in that the microorganisms used are Hanseniospora guillermondii, Pichia anomala and Pichia jadinii . 3. Procedure for the production of conjugated linoleic acids characterized by the following stages:

to)

incubation of a microorganism producing conjugated linoleic acids, to be chosen from the group consisting of bacteria, yeasts and fungi, preferably non- Saccharomyces yeasts, in liquid medium, in the presence of vaccenic acid; Y

b)

linoleic acid extraction conjugates resulting from the incubation of step a); purification and characterization of said acids.

4. Method of production of conjugated linoleic acids according to claim 3, characterized in that the microorganisms used are the yeasts Hanseniaspora guillermondii, Pichia anomala , and Pichia jadinii . 5. Method of production of conjugated linoleic acids according to claims 1-4, characterized in that the conjugated linoleic acid obtained is cis-9-trans-11-octadecadienoic acid. 6. Process for the production of baking yeast biomass and bread enriched in conjugated linoleic acids, characterized in that it consists in adding to the bread dough baker's yeast loaded with the cis-9-trans-11 isomer of conjugated linoleic acid at a concentration of 1% 3% by weight following step a) of any of claims 1 to 4. 7. Procedure for the production of yogurt enriched in conjugated linoleic acids, characterized in that it consists in adding to the fermentation medium a yogurt mixture loaded with the cis-9-trans-11 isomer of the conjugated linoleic acid at a concentration 0.2% to 2% by weight following step a) of any of claims 1 to 4.