ITRM20090662A1 - PROBIOTIC BACTERIA STRAINS CONJUGATE MANUFACTURERS OF LINOLEIC ACID - Google Patents

Description

Strains of probiotic bacteria producing Linoleic Acid Conjugates.

DESCRIPTION

The present invention relates to strains of probiotic bacteria producing linoleic acid (CLA) conjugates. In particular, the present invention refers to a selection of strains of bacteria belonging to the genus Bifidobacterium which have been selected for their ability to produce conjugates of linoleic acid (CLA), from linoleic acid (LA). Furthermore, the present invention relates to a method for in situ production of linoleic acid derivatives. Finally, the present invention relates to a food, dietary or pharmaceutical composition comprising said strains of bacteria capable of increasing the quantity of CLA in situ, i.e. inside the gastrointestinal tract.

It is known that in recent years there has been an increasing use of conjugates of linoleic acid (CLA) in the preparation of foods, supplements and drugs.

The development of these preparations is linked to the well-known beneficial effects carried out by CLA within the body. In particular, among the beneficial effects recognized to CLA there is an anti-inflammatory effect / activity. Additional beneficial actions reported are the increase in lean mass, the increase in thermogenesis, cancer prevention and protection against oxidative stress.

Over the past three decades, however, dietary CLA intake has dropped dramatically for two basic reasons. Firstly, following the wave of health that has affected the entire Western world, the consumption of pork and milk derivatives, the main sources of these molecules, has decreased considerably. Secondly, modern livestock farming techniques have gradually led to the replacement of grassland grasses with industrially produced feeds which are not always rich in natural linoleic acid.

Considering that the intake of CLA with a normal diet has proved insufficient, and moreover it is not even entirely desirable to increase the consumption of foods such as meat or dairy products, it is more advisable to introduce CLA in alternative forms.

Therefore, the growing interest in the use of CLA in the nutrition sector and in the nutraceutical and pharmaceutical fields has led to the development of methods for the production of linoleic acid conjugates, in particular by microbial route.

Concerning the microbial production of CLA, many efforts have been made to optimize processes for the production of CLA from microorganisms and linoleic acid.

The main objective of these production processes was the use of CLA produced to prepare new foods in which CLA is added to a food substrate to prepare foods rich in CLA.

However, said food products added with CLA are not without drawbacks and have some limitations deriving from the fact that their oral administration does not always involve a real and effective localization of CLA in the gastrointestinal tract of interest.

Furthermore, in some countries the direct addition of CLA as an active ingredient in food products is not allowed.

Therefore, it is necessary to have a composition capable of overcoming the limitations of the known art and that is able to provide CLA directly in the body, for example in the gastrointestinal tract of interest.

Furthermore, linoleic acid, in excessive quantities, is known to be harmful to human health. Linoleic acid, in fact, is a ω-6 fatty acid, and it is known that an excess of ω-6 acids compared to ω-3, a situation typical of Western societies, is responsible for a series of negative effects, including : increased allergic and inflammatory reactions, blood pressure, platelet aggregation and, consequently, cardiovascular risk. An excessive intake of omega-6 also interferes with the formation of omega-3s.

Microorganisms capable of converting linoleic acid into CLA therefore seem to adopt a detoxification strategy by transforming said acid into its conjugates and thus removing it from the environment in which it is found.

The excess of linoleic acid in the modern human diet is not due exclusively to a low consumption of fish but, rather, also to a high consumption of products rich in vegetable and seed oils. Moreover, this situation is not only characteristic of the human diet but, on the contrary, it also extends to the diet of feeding animals which are commonly fed on grains.

All this is reflected, consequently, in a worse ω-6 / ω-3 ratio even in meat, dairy and egg products which are among the most consumed foods at the table. Furthermore, an improvement in the ω-6 / ω-3 ratio by dietary means presents today some difficulties and problems, which are difficult to overcome, as they are linked to the imposed production and marketing systems managed on an industrial scale that are difficult to modify.

Therefore, it is necessary to have a composition capable of converting linoleic acid (LA), deriving from the diet and which accumulates in excess in the body, into conjugates of linoleic acid (CLA), in such a way as to achieve effective on-site production.

In particular, it is necessary to have a composition comprising probiotic microorganisms capable of being able to overcome the gastric barrier and duodenal transit, to colonize the gastrointestinal tract and to be able to convert LA into CLA directly in situ, preferably in the gastrointestinal tract.

An object of the present invention forms new strains of probiotic bacteria capable of converting LA into CLA, as claimed in the accompanying claim.

A further object of the present invention is a food or dietetic or pharmaceutical composition, as claimed in the attached claim.

A further object of the present invention forms a pharmaceutical composition, as claimed in the attached claim.

A further object of the present invention is a method for the production of CLA, as claimed in the attached claim.

A further object of the present invention is the use of at least one bacterium strain for the preparation of a composition, as claimed in the accompanying claim.

Further preferred embodiments of the present invention will be reported and illustrated below without wishing to limit in any way the scope of the present invention.

Table 1 shows the qualitative and quantitative composition of the three cultural media used in the present invention: TPY, MRS and LATPg.

Table 2 reports the absorbance values obtained following the spectrophotometric reading at wavelength 233 nm with respect to the CLA concentrations.

Table 3 shows the regression line obtained with the values shown in table 2.

Table 4 reports plate counts for inclusion using TPY and MRS media at different LA concentrations.

Table 5 reports the obtained concentration values of CLA / ml produced; percentage of conversion of LA to CLA (expressed as CLA products / 0.50 mg / ml LA); concentration ratio CLA / OD 600 nm.

Table 6 reports the quantification of CLA present constitutively in TPY medium with and without LA inoculation.

Table 7 reports the resistance to gastric juices, bile and pancreatic secretion of the selected strains.

Figure 1 shows the growth curves over time of strain B. short DSM 20213 in TPY and MRS broth at different concentrations of LA (0, 0.5 and 1 mg / ml). The initial pH for TPY broth cultures is 6.11; in MRS it is 5.83. The final pH values are shown in the figure itself.

The Applicant, following an intense research activity, has come to select specific strains of probiotic bacteria, belonging to the genus Bifidobacterium.

Advantageously, said strains belong to the B. longum, B. breve, B. lactis and B. bifidum species.

In particular, the strains selected by the Applicant are selected from the group comprising:

- Short Bifidobacterium, filed with the DSMZ and having access number DSM 16604, filing date 20.07.2004, owner Probiotical S.p.A .;

- Short Bifidobacterium, filed with the DSMZ and having access number DSM 16596, filing date 21.07.2004, owner Probiotical S.p.A .;

- Bifidobacterium breve, filed with the DSMZ and having access number DSM 20213.

The selected strains are capable of converting linoleic acid (LA) into conjugates of linoleic acid (CLA), with a degree of conversion greater than 65%.

In the context of the present invention, by CLA is meant the group comprising the conjugates cis-9, trans-11 CLA and trans-10, cis-12 CLA.

All the strains tested by the Applicant are of human origin and have been isolated from fecal material.

In addition, the selected strains were also tested for their resistance to gastric juices, bile and pancreatic secretion.

The results are reported in table 7.

In table 7, (^) indicates the survival of the probiotic strains in two different types of gastric juices and in a simulated pancreatic secretion at 37 ° C after different contact times (5, 30 and 60 minutes).

In table 7, (^^) indicates the survival results in the presence of bile secretion by comparing the number of colonies grown in a medium "with" and "without" the addition of bile salts or human bile.

From these tests it emerged that a significant percentage of ingested cells is able to overcome the gastric acidity barrier and overcome the intestine.

This aspect is of particular importance as it ensures that a sufficient number of viable cells is able to reach the intestine, where it mediates the transformation of linoleic acid into its conjugates.

In a preferred embodiment, the food or dietetic or pharmaceutical composition of the present invention comprises at least one strain belonging to the B. short species, preferably two or three strains.

Advantageously, the composition comprises:

- Short Bifidobacterium, filed with the DSMZ and having access number DSM 16604, filing date 20.07.2004, owner of Probiotical S.p.A., and / or

- Short Bifidobacterium, filed with the DSMZ and having access number DSM 16596, filing date 21.07.2004, owner of Probiotical S.p.A., and / or

- Bifidobacterium breve, filed with the DSMZ and having access number DSM 20213.

In case of use of two B. short species, the ratio in viable cells ranges from 1: 3 to 3: 1, preferably 1: 1.

Advantageously, the composition comprises Bifidobacterium breve DSM 16604 and Bifidobacterium breve DSM 16596.

Advantageously, the composition comprises Bifidobacterium brevis DSM 16604, Bifidobacterium brew DSM 16596 and Bifidobacterium brew DSM 20213.

In a preferred embodiment, the pharmaceutical composition containing the strains of bacteria as specified above is indicated for use as a medicament, preferably as an anti-inflammatory, in particular for the preventive and / or curative treatment of intestinal disorders, diarrhea, inflammation of the colon, increase in mass lean, increased thermogenesis, cancer prevention and protection against oxidative stress.

The composition of the present invention finds application in the preventive or curative treatment of a deficiency of conjugates of linoleic acid.

The composition of the present invention can be formulated in solid, lyophilized or dried form, for example in powder or granules.

Also, a pharmaceutical form of interest is the hard or soft tablet or capsule.

As far as the tablet is concerned, this may comprise an internal part comprising the strains of bacteria and an external lining part. The coating may comprise water-soluble polymers and / or polymers capable of resisting changes in stomach pH and allowing passage into the intestinal tract.

Another aspect of the invention is directed to a method for the production of conjugates of linoleic acid.

The method involves a phase in which one or more strains of bacteria, as specified above, are cultured / fermented in the presence of linoleic acid and, subsequently, the conjugate of the linoleic acid formed is isolated.

The method can be carried out in the laboratory or on an industrial scale.

In another aspect of the invention the conversion from LA to CLA can take place directly in the organism once the individual has assumed the composition of the present invention.

The conjugate of linoleic acid that is obtained in greater quantities is cis-9, trans-11 octadecadienoic acid.

Another aspect of the invention is directed to the use of at least one bacterium strain, as specified above, for the preparation of a composition for the treatment of disorders or pathologies connected to a deficiency of linoleic acid derivatives.

In a first experimental phase, the Applicant endeavored to develop the best choice of the culture medium suitable for conducting the subsequent tests which concerned the development of spectrophotometric techniques, with reading at a wavelength of 233 nm, which can be used for make a selection of CLA-producing strains of bacteria.

Linoleic acid toxicity test and choice of culture medium.

Strain B. short DSMZ 20213 was selected as a candidate for the linoleic acid toxicity test. This strain, in fact, is able to grow in the presence of LA and has good conversion rates to CLA. In addition, B. short DSMZ 20213 was used for subsequent analyzes as a positive control. Therefore, starting from the frozen form of said strain, it was therefore possible to revitalize it in three different cultural means which are explained in Table 1.

After preparation, the media were autoclaved at 121 ° C for 15 '. At the end of sterilization, the pH of the media were 6.60 ± 0.10 at 25 ° C, 6.20 ± 0.20 at 25 ° C and 6.5 ± 0.5 at 25 ° C for TPY, MRS and LAPTg broth. The revitalization was performed by inoculating 1% of B. short DSMZ 20213 in 10 ml of the three culture media with the addition of 1% of cysteine hydrochloride (sol. 5%) and subsequent incubation in anaerobiosis with Gas-Pack at 37 ° C ± 1 ° C for 24 hours ± 1 hour. This operation was performed by two successive transplants to allow the full reactivation of the strain.

The revitalized strain was inoculated at 1% in 10 ml of the three different media prepared as above with the addition of three solutions at different concentrations of linoleic acid and, to be precise, at 0.5 mg / ml (Sigma- Aldrich code L1276) and then incubated in the Gas-Pack at 37 ± ° C for 16 hours ± 1 hour.

To simplify the operation, three stock solutions of LA were produced at 0 mg / ml, 50 mg / ml and 100 mg / ml with the addition of 2% (v / v) of Tween 80 (to allow the formation of an emulsion with consequent increase in the solubility of dissolved linoleic acid through the use of a stir bar and a magnetic stirrer.

When the Tween 80 was completely dissolved, the solutions thus obtained were aliquoted and frozen at -25 ± 1 ° C after filtration with a filter at 0.20 μm.

Counting by spectrophotometric reading at 600 nm was performed at the beginning, at 6 hours and at the end of the incubation period. The pH was recorded at the beginning and at the end of the incubation period.

Preparation of the regression line

The regression line constructed for CLA c9 / t11 concentrations equal to 320, 160, 80, 40, 20 and 10 μg / ml hexane, showed that a linear increase in absorbance values corresponds to concentrations from 10 to 80 μg / ml. at 233 nm (R2 = 1).

Instead, by calculating the regression line also with the absorbance value obtained for the concentration of 160 μg / ml, the line loses the linear trend and becomes polynomial not allowing, in fact, a precise quantification of the CLA concentrations for absorbances between 2.095 and 2.733. At the concentration of 320 μg / ml and above, the spectrophotometer gave non-quantifiable absorbance (> 3).

Therefore, the CLA concentration present in the culture supernatants of absorbance <2.095 can be calculated directly with the equation y = 25.311x + 0.0698. For samples that gave readings greater than 2.095, the expedient of serial dilutions 1: 2 was used.

By resorting to the serial dilution of the sample 1: 2 we have therefore brought back the absorbance values in the interval that guarantees the linearity and, therefore, the applicability of the equation. The data are shown in Table 2.

To overcome the difficulty in dosing CLA c9 / t11 (in the preparation of the first dilution necessary for the calculation of the regression line) due to its viscosity, and to ensure maximum precision, we used a fixed volume pipette with glass capillaries to be 2 μl.

The 2 μl sample of CLA c9 / t11 thus obtained was then diluted with 1,450 ml of hexane in a 2 ml tube.

We therefore obtained a starting concentration of 1280 μg / ml (density c9 / t11 = 0.903 mg / μl). From this concentration, serial dilutions 1: 2 were made up to a concentration of 10 μg / ml. To limit the evaporation of the hexane as much as possible, the various serial dilutions were carried out in 200 μl microtubes previously filled with 100 μl of hexane and then immediately closed again. The various dilutions were then obtained serially by adding 100 μl of the previous solution, starting from the stock solution at 1280 μg / ml.

At the end of the preparation of the various dilutions, we carried out the reading of the optical density with the spectrophotometer at the wavelength of 233 nm using quartz couvettes with a nominal capacity of 100 μl. This operation was performed starting from the solution with the lowest concentration (10 μg / ml).

The absorbance values thus obtained have been reported in table 3.

Extraction of fatty acids.

After choosing the suitable culture medium and the concentrations of LA to use, they were thawed and reactivated.

Strain B. short DSMZ 20213 was selected as the positive control. The same procedure was also applied to a sample of soil without inoculation and with the addition of LA to be used as a blank.

TPY broth, after being added with 1% cysteine hydrochloride (sol. 5%), was chosen as a culture medium for the reactivation and growth phases in the presence of LA at 0.5 mg / ml.

At the end of the incubation period, the broths and the blank were centrifuged at 5000g for 5 ́ and the supernatants were aliquoted at the rate of 500 μl in 1.5 ml microcentrifuge tubes.

Therefore, following a double extraction with hexane, according to the methodology of Yung et al., 2006 (Reference 1) with the following modifications. The extraction involved adding 500 μl of hexane to the aliquoted supernatants and the blank. The samples were then mixed by inversion for 10 ', taking care not to break the interface between the two phases and then left to stand for 30' at room temperature. At the end of the resting phase, the samples were centrifuged at 2000 g for 5 ́ to facilitate the sampling of the upper phase in hexane. Once the hexane phase was withdrawn and stored in a 2 ml tube, the lower phase was again treated as above with 500 μl of hexane. The upper phase was added to the previous one and mixed by tilting.

Spectrophotometric analysis.

The quantification of CLA produced was performed by a spectrophotometer optical density reading using a wavelength of 233 nm according to Barrett et al., 2007 (Reference 2) and Xu et al., 2008 (Reference 3).

Before carrying out the analysis, the spectrophotometer was zeroed against 100 μl of hexane using a quartz cup.

The reading was then carried out for all the samples starting from the blank, taking care to prime the couvette with hexane before proceeding with the transfer of the sample. For the samples that gave absorbance values> 3, serial dilutions 1: 2 were carried out in hexane up to the absorbance values which allowed the calculation of the CLA concentrations by interpolation of the straight line Y = 25.311 x 0.0698 .

The spectrophotometric techniques for the quantification of CLA produced by bacterial cultures are known from Barrette et al., 2007. This spectrophotometric technique is based on the characteristic of conjugated dienes (in this case CLA) to possess a UV spectrum typical of this class of compounds, with an absorbance of 233 nm. This method, however, allows the quantification of CLA produced.

Linoleic acid toxicity test and choice of culture medium.

At the end of the incubation period (16 hours ± 1 hour, 37 ± 1 ° C), in the toxicity test conducted on the B. short strain DSM 20213 using different concentrations of LA (0, 0.5 and 1 mg / ml) in three different soils (TPY, MRS and LAPTg) made it possible to identify the most suitable culture medium to conduct the subsequent screening phase. TPY broth was chosen as a culture medium because it dictated the environmental conditions, in the presence of LA, more favorable to the growth of Bifidobacteria. With the TPY broth the lower count variability was found both by reading the optical density at the spectrophotometer with a wavelength at 600 nm and by counting on the inclusion plate, figure 1 and table 4.

The differences observed in the use of TPY, MRS and LAPTg broth at different concentrations of LA (0, 035, 1 mg / ml) (figure 1) underlined the importance of the culture medium in studies concerning microbial metabolism.

In fact, the use of TPY broth was found to be preferable compared to the use of MRS and LAPTg broth giving the lowest variability of microbial growth, at the different concentrations of LA, as well as the higher counts, with the same concentration of LA (0.5 and 1 mg / mL), compared to RMS and LAPTg broth. The initial chosen concentration of LA used was 0.5 mg / ml. This concentration is, in fact, the preferable one for the LA to CLA conversion tests in broth MRS conducted in other studies (Barrett et al., 2007 and Xu et al., 2008). The choice was dictated by the desire to compare, in the preliminary phase, the conversion rates using the TPY broth culture medium and those present in the literature concerning the use of the MRS broth culture medium.

Selection of CLA producing bifidobacteria strains.

At the end of the incubation period (16 hours ± 1 hour, 37 ± 1 ° C), the above bacterial strains and the negative control Lb. Reuteri DSM 20016 underwent the procedure for spectrophotometric analysis at 233 nm necessary for the quantification of CLA products. Table 5 shows the results obtained relating to the conversion of LA to CLA by the various strains of selected bifidobacteria.

The B. short DSM 16604 strain was found to be the major producer of CLA showing a production of 0.393 mg / ml equal to a conversion rate from LA to CLA of 71.38%.

The concentrations of CLA produced were calculated by assuming that the culture media initially contained variable quantities of LA and CLA due to the presence of ingredients such as meat extracts, yeast extract, soy components, etc. in this regard, we compared the optical density readings at the spectrophotometer at the wavelength of 233 nm, of the different sterile media with and without LA at 0.5 mg / ml.

From the results obtained (table 6) no significant difference was found between the sterile medium without and with the addition of 0.5 mg / ml of LA.

The above confirmed the ability of the technique used to quantify only the acidic component characterized by having two conjugated double bonds such as CLA. Finally, the calculated amount of CLA present in sterile soils was subsequently subtracted in the calculation of the concentrations of CLA produced by microbial activity. Furthermore, the bacterial count carried out by reading the optical density at the spectrophotometer (wavelength 600 nm) for the L. reuteri DSM 20016 showed a higher count compared to the producing strains suggesting that the toxicity related to the presence of LA is strain specific.

Based on these results, it can be deduced that the most sensitive microorganisms are also those capable of carrying out the greatest conversion of LA to CLA, probably as a defense mechanism against the toxic element.

The tests performed above show a high percentage of conversion obtained for the B. short DSM 16604 (71.38%) compared to the positive control B. short DSM 20213 relative to a work previously carried out with the use of MRS broth (Barrett et al ., 2007).

Therefore, the conversion activity from LA to CLA by Bifidobacteria could make this metabolite available directly in the intestinal lumen, and then manifest its health properties both locally and following its absorption.

The above shows the ability of some strains of bifidobacteria (in particular B. short) to convert linoleic acid into conjugates of linoleic acid. Therefore, the use of these strains in the probiotic field could represent an effective and innovative way to control the delicate balance between AGE omega-6 / omega-3 taken through a diet.

Bifidobacteria strains capable of converting LA to CLA could interact in situ, along the gastrointestinal tract, with the essential fatty acid component taken through the diet, potentially counteracting the pro-inflammatory metabolism of the omega-fatty acid pathway. 6 and producing metabolites (CLA) with possible nutraceutical role. In fact, even though most of the fat consumed in the diet is digested in the small intestine, approximately 5-8 g of lipids arrive in the colon each day. In addition, it has been estimated that humans excrete about 20 mg of LA via the faeces. This suggests that LA is potentially usable as a substrate for CLA production. In addition, the recent isolation of bifidobacteria and lactobacilli strains from the small intestine of infants extends the applicability of CLA-producing strains of Bifidobacteria. These findings suggest an extended field of applicability in almost the entire gastrointestinal tract, allowing in fact a potentially simultaneous action to the lipid absorption that occurs mainly in the upper regions of the small intestine. Finally, the applicability of CLA-producing probiotic strains seems promising, not least because the anticarcinogenic effect of CLA could occur at very low concentrations. In fact, anticarcinogenic effects were observed in animal models for a quantity of CLA equal to 0.5 - 1% (w / w) of the daily diet.

LIST OF REFERENCES

1) M. Y. Young et al., Technical Note: Improved Extraction Method with Hexane for Gas Chromatograpic Analysis of Conjugated Linoleic Acids, J. Diary Sci. 89: 90-94, American Diary Science Association, 2006.

2) E. Barrett et al., Rapid Screening Method for Analyzing the Conjugated Linoleic Acid Production Capabilities of Bacterial Cultures, Applied and Environmental Microbiology, Apr. 2007, p. 2333 - 2337, American Society for Microbiology.

3) H. Xu et al., Kinetics of microbial hydrogenation of free linoleic acid to conjucated linoleic acids, Journal of Applied Microbiology, ISSN 1364-5072.

Claims (10)

CLAIMS 1. A probiotic bacterium strain belonging to the genus Bifidobacterium selected from the group comprising: Bifidobacterium breve, filed with the DSMZ on 20.07.2004 and having access number DSM 16604, and Bifidobacterium breve, filed with the DSMZ on 21.07.2004 and having access number DSM 16596. 2. A probiotic bacterium strain belonging to the genus Bifidobacterium selected from the group comprising: Bifidobacterium breve, filed with the DSMZ on 20.07.2004 and having access number DSM 16604, Bifidobacterium breve, filed with the DSMZ on 21.07.2004 and having access number DSM 16596, and Bifidobacterium breve, filed with the DSMZ and having DSM access number 20213, said strain being able to convert linoleic acid into conjugates of linoleic acid; preferably said conversion is greater than 65%. A food or dietary or pharmaceutical composition comprising at least one strain of bacteria according to claim 1 or 2. The pharmaceutical composition according to claim 3, for use as a medicament; preferably as an anti-inflammatory. 5. The pharmaceutical composition according to claim 3, for use in the preventive or curative treatment of a deficiency of linoleic acid conjugates. The composition according to one of claims 3-5, wherein said composition comprises Bifidobacterium breve DSM 16604 and Bifidobacterium breve DSM 16596. The composition according to one of claims 3-6, wherein said composition comprises two species B. short, in a ratio in viable cells is comprised from 1: 3 to 3: 1; preferably 1: 1. 8. A method for the production of linoleic acid conjugates by culturing a bacterium strain selected according to claim 2 in the presence of linoleic acid and isolating the formed linoleic acid conjugate. 9. The method according to claim 8, wherein the obtained linoleic acid conjugate is cis-9, trans-11 octadecadienoic acid. 10. Use of at least one bacterium strain according to claim 2, for the preparation of a composition for the treatment of disorders or pathologies connected to a deficiency of linoleic acid derivatives or of a composition with anti-inflammatory activity.