WO2006035107A1

WO2006035107A1 - Process for esterifying fatty acids

Info

Publication number: WO2006035107A1
Application number: PCT/FI2005/000411
Authority: WO
Inventors: Simo Laakso; Marjatta Vahvaselkä; Sanna Jaakola; Pekka Lehtinen
Original assignee: Raisio Oyj
Priority date: 2004-09-27
Filing date: 2005-09-27
Publication date: 2006-04-06
Also published as: FI20041240A0

Abstract

The invention relates to a process for esterifying fatty acids added to edible preparations. The process and the product in accordance with the invention are characterised by a fatty acid being added to or produced in a mixture allowing the culture of eucaryotic micro-organisms, the obtained micro-organism mass as such or its components being used as an edible preparation.

Description

PROCESS FOR ESTERIFYING FATTY ACIDS USING MICROORGANISMS

The invention relates to a process for esterifying fatty acids to the lipids of eucaryotic micro-organisms and to the use of mixtures containing such eucaryotic micro-organisms or their parts as an edible product or as part of an edible product.

In food materials in a natural state, free fatty acids account for only a minuscule portion of the fat contained in the material. Thus for instance, free fatty acids account for about 2-11 % of the total fat in corn immediately after the crushing of the grains (Zhou, M., Robards, K., Glennie-Hahnes, M., Helliwell, S., 1999, Oat Lipids, JAOCS 76(2), 159-169). Should the proportion of free fatty acids in foodstuffs be substantially higher than this due either to an addition or to rancidness, this appears as a flaw that can be sensed.

On the other hand, there is a strongly increasing interest in raising the content of fatty acids having a beneficial health effect in foodstuffs, such as e.g. fatty acids of conjugated linolenic acid (CLA) or omega-3 fatty acids. Conjugated linolenic acid is obtained from natural oil by concentrating e.g. using water steam distillation, however, when a desired isomer structure is aimed at, the preparation takes place by isomerising free linolenic acid. In addition to causing a bitter taste in the foodstuff, fatty acids in the form of free acids containing carbon-carbon double bonds are susceptible to oxidation and thus also increase the oxidative susceptibility of the other fats in the nutrient. The use of free fatty acids as an ingredient in foodstuffs may even be a health hazard. Free fatty acids also affect the physical properties of the fat contained in the foodstuff, such as its melting point and the structure of the foodstuff, especially if the free acid addition replaces other fats in the foodstuff. Esterification of the free acid to form a part of triacylglycerol (i.e. triglyceride) or polar lipids (e.g. phospholipids and glycolipids) would eliminate the problems described above.

In fact, the problems relating to additions of free fatty acids have been identified, and in order to add fatty acids with beneficial health effects without the perception and other problems described above, methods have been described, in which the fatty acid is esterified to compounds having an alcohol group. Esterification can be performed by conventional basic chemical processes or lipase enzymes using reaction conditions favouring the formation of an ester bond instead of ester hydrolysis. Enzymatic esterification and conditions necessary for this are described i.a. in the references Haas, M. J., Kramer, J. K. G., McNeill, G., Scott, K., Foglia, T. A., Sehat, N., Fritsche, J., Mossoba, M. M., Yurawecz, M. P., 1999, Lipase- Catalyzed Fractionation of Conjugated Linolenic Acid Isomers, Lipids 34(9), 979- 987 and McNeill, G. P., Rawlins, C, Peilow, A. C, 1999, Enzymatic Enrichment of Conjugated Linolenic Acid Isomers and Incorporation into Triglycerides, JAOCS 76(11 ), 1265-1268, 1-6, in which CLA was the esterified fatty acid. The catalyst consisted e.g. of lipase isolated from the mould Geotrichυm candidum.

However, known methods used for esterifying fatty acids, such as CLA, involve substantial shortcomings and restrictions when used in foodstuffs. Chemical esterification requires the use of non-polar solvents and an acid catalyst and also a raised temperature. In the esterification of CLA, for instance, such a process produces a mixture of several CLA isomers. Also, the addition to a foodstuff of a fatty acid ester produced by chemical means in an organic solvent environment will entail time-consuming and costly approval procedures. In addition, a chemically esterified fatty acid, such as a nutrient supplemented with CLA, does not represent the concept of a natural foodstuff. Thus, the use of chemical esterification is at least not adequate in the esterification of free CLA prepared by natural methods, such as microbiological isomerisation of linolenic acid.

Esterification performed with the aid of lipases also comprises steps that involve problems in terms of using the product as a foodstuff. The lipase catalysis also requires the use of non-polar solvents. Moreover, commonly available lipases derive from microbes, and hence their production processes have not been documented and received approval for use as foodstuffs.

The use of known esterification techniques have the common feature of not being suitable as part of a conventional food production process.

Consequently, there is an obvious need for esterifying fatty acids with beneficial health effects added to foodstuffs and especially for using to this end a process, whose all partial steps represent natural and conventional food processing and thus produce products that are safe when used as foodstuffs and will pass regulatory approval procedures. According to the invention there is provided a process for modifying a fatty acid ester composition in an edible product, with at least a portion of the fatty acid esters converted into a fatty acid ester, whose fatty acid residue contains a fatty acid residue having beneficial health effects, the process comprising the following steps

- a free fatty acid having beneficial health effects is added to or produced in a mixture containing nutrients necessary for an eucaryotic micro-organism,

- an eucaryotic micro-organism is inoculated in the mixture containing fatty acid,

- the eucaryotic micro-organism is cultivated in order to produce a microbial mass on the substrate, the free fatty acid having beneficial health effects migrating to the cell and being esterified under the action of the eucaryotic micro-organism to fatty acid ester and

- the microbial mass containing fatty acid esters thus obtained is added to the edible product or is used as an edible product.

At least a portion of the fatty acid esters is preferably a triglyceride, in which 1-3 acyl groups is a residue of said fatty acid having beneficial health effects.

The fatty acid having beneficial health effects is preferably a conjugated linolenic acid, an omega-3 or omega-6 fatty acid or a mixture of these.

Conjugated linolenic acid is a particularly preferred fatty acid with beneficial health effects.

The conjugated linolenic acid can be obtained by isomerisation of linolenic acid derived from corn, such as oat, for instance.

The mixture used for cultivating the eucaryotic micro-organism may contain corn.

The eucaryotic micro-organism is a food-grade micro-organism, preferably food- grade yeast.

Said yeast may pertain to the genii Saccharomyces, Kluyveromyces or Candida, e.g. Saccharomyces cerevisiae or Kluyveromyces marxianus (Candida kefyr).

Under the esterification conditions, the eucaryotic micro-organism is preferably incapable of isomerising the fatty acid to another fatty acid. Said microbial mass and the substrate can be added to the edible product or used as an edible product.

Said microbial mass or alternatively the microbial mass and the substrate can also be dried and added to the edible product or used as an edible product.

According to the invention there is also provided an edible product containing an eucaryotic micro-organism, in the cells or cell parts of which the esterified fatty acids have a composition such that the proportion of the fatty acid residue having beneficial health effects has been increased, or a fatty acid residue having beneficial health effects has been formed in the composition.

The edible product modified in accordance with the invention may originally contain esterified fatty acids containing fatty acid residues with beneficial health effects, the proportion of fatty acid residues with beneficial health effects being increased in accordance with the invention. The edible product modified in accordance with the invention may also be such that does not originally contain esterified fatty acids containing fatty acid residues with beneficial health effects, and then fatty acid residues having beneficial health effects are produced in accordance with the invention.

The fatty acid residue having beneficial health effects in the edible product of the invention is preferably a conjugated linolenic acid.

The prime novelty of the described invention is the use of entire eucaryotic microbe cells instead of isolated lipase enzymes for binding CLA or any other fatty acid having beneficial health effects that is in free acid form in the substrate to form part of the acyl lipids in the cell and the use of such eucaryotic micro¬ organisms for the binding whose use in food production and as food ingredients has been previously approved and established. The desired free fatty acid is brought in a mixture, in which food-grade eucaryotic micro-organisms, such as yeast, can be cultivated, the substrate thus obtained as such, the isolated microbial mass, or fractions isolated from the mass, such as crushed cells, being usable for increasing the concentration of the desired fatty acid ester form in the edible product. Eucaryotic micro-organisms are preferably cultivated in a nutrient medium containing a minimum of fatty acids other than the desired ones and/or in substrates that can be added as such to the desired food, without cell separation. The process is particularly advantageously applied to a process, where free CLA has been prepared by micro-biological isomerisation of linolenic acid, because the culture of eucaryotic micro-organisms esterifying CLA can be carried out in the same substrate as the preceding isomerisation.

The choice of the eucaryotic micro-organism used for esterification has a great impact on the yield of fat in ester form and on the fat composition. In the process of the invention, one preferably uses eucaryotic micro-organisms, which have been previously used in food production or occur naturally in conventional foodstuffs. Yeasts, such as baking yeast, have conventionally been used in food production and as a food component. Yeast is inexpensive and yeast culture represents straightforward and known techniques. As eucaryotics, yeasts are capable of synthesising and storing also neural lipids in their cells. Yeasts have a relatively high fat content, in the range 12-14% for baking yeast, for instance, and in some yeast strains the fat accumulation may account for up to 65% of the total dry weight of the cell. Yeast additions to a foodstuff may consequently achieve high concentrations of added fatty acid.

The process avoids the major inconveniences relating to prior art esterification techniques, such as the use of organic solvents, acid catalysts and enzymes derived from mould. The esterification of the invention can be implemented using simple devices, it can be used even in large-scale production and in any other food production without special premises.

Yeast poses low requirements on the substrate. The use of yeast is thus suitable for esterification of free CLA produced by microbial isomerisation. Such processes for yielding free CLA have been described i.a. in the references:

Jiang, J., Bjόrck, L., Fonden, R., 1998, Production of conjugated linolenic acid by dairy starter cultures, J. Appl. Microbiol. 85, 95-102 and Vahvaselka, M., Lehtinen, P., Sippola, S., Laakso, S., 2004, Enrichment of conjugated linolenic acid in oats (Avena sauva L.) by microbial isomerization, J. Agric. Food Chem. 52, 1749-1752. In this conjunction, yeast can be cultivated at the same time as another organism performs isomerisation of linolenic acid, or yeast can be added to the same substrate after isomerisation. The amount of yeast cells in the process of the invention may preferably rise to an approximate value of 2 - 4 mg of dry substance per ml of substrate. In this case, 0.2 - 0.4 mg/ml of CLA is preferably added to the substrate for esterification of all the free CLA. If the cell yield differs from the amounts above, the CLA content of the substrate can be adjusted to a higher or lower value, respectively.

The process of the invention provides products usable as such in foodstuffs, such as yeast. Alternatively, the substrate used for esterification of fatty acid may contain other food components, such as corn, and then a cereal is obtained which contains the desired fatty acid in ester form.

The fat content of yeasts containing esterified CLA or the degree of unsaturation of the fat do not change substantially, and, thus, the yeast produced in accordance with the invention is usable for their established purposes of use, without their physical characteristics changing.

The invention is explained in further detail below by means of examples of preferred embodiments and figures.

Figure 1 illustrates the effect of the CLA content of the substrate on the fatty acid composition of the yeast cells Saccharomyces cerevisiae.

Figure 2 illustrates the fatty acid compositions of the lipid classes of the yeast cells S. cerevisiae when the substrate contains 0.2 mg/ml of CLA. TG is triacyl glycerol, PL denotes polar lipids.

Figure 3 illustrates the effect of alpha or gamma linolenic acid (0.2 mg/ml) in the medium culture on the fatty acid composition of the yeast cells S. cerevisiae.

Figure 4 illustrates the effect of alpha or gamma linolenic acid (0.2 mg/ml) in the medium culture on the lipid class composition of the yeast cells S. cerevisiae.

Figure 5 illustrates the effect of CLA (0.2 mg/ml) in the substrate on the fatty acid composition of the yeast cells Kluyveromyces marxianus. LA stands for linolenic acid. Figure 6 illustrates the effect of the CLA (0.2 mg/ml) in the substrate on the fatty acid composition of the lipid classes of the cell yeasts K. marxianus.

Example 1

Conjugated linolenic acid (CLA) was added in various concentrations to a baking yeast substrate and the yeast was cultivated for 17 hours in order to produce a cell mass. An identical culture without CLA addition was used as a control. The CLA did not affect the yeast growth.

When the substrate contained 0.2 - 0.4 mg/ml of conjugated linolenic acid, it was observed that the CLA proportion in the total fat isolated from the yeast (10.1 w% of the dry weight of the cells) was 47 molar % of all the fatty acids (figure 1). The CLA proportion both in polar lipids and in triglycerides was 39 molar % of all the fatty acids (figure 2). Figure 1 also shows that, compared to the fats of the control culture yeast, the amounts of palmitoleic acid and oleic acid had decreased. Using yeast, free CLA was thus esterified and its proportion was markedly increased in yeast triglycerides and polar lipids.

The tests used the strain Saccharomyces cerevisiae VTT-72021. The yeast strain was cultivated in a Worth broth (base) (Merck) at a temperature of 30 ° for 17 hours, agitation at 270 rpm. 1 ml of this cell culture was inoculated in 50 ml of identical culture broth, to which CLA (Sigma) had been added in varying amounts. The test also included a control culture to which no fatty acids had been added. The cells were cultivated in two parallel Erlenmeyer flasks at a temperature of 30 ⁰C during agitation (270 rpm) for 17 hours. Then they were centrifuged for separation at a temperature of 4 °C for 15 min (6,000 rpm), followed by washing with cold tap water, repeated centrifugation as above and cold drying.

CLA migration to the yeast fats was examined by extracting the fats with a mixture of dichloromethane-methanol and by separating the lipid classes to polar lipids, triglycerides and free fatty acids by thin layer chromatography (Liukkonen, K.H., Montfoort, A., Laakso, S.V., 1992, Water-induced lipid changes in oat processing, J. Agric. Food Chem. 40, 126-130). The fatty acid composition and concentration of each lipid class was determined by gas chromatography (Suutari, M., Liukkonen, K., Laakso, S., 1990, Temperature adaptation in yeasts: the role of fatty acids, J. Gen. Microbiol. 136, 1469-1474). Example 2

The yeast Saccharomyces cerevisiae (VTT-72021) was cultured in the manner described in example 1. Instead of CLA₁ 0,2 mg/ml of either alpha or gamma linolenic acid (Sigma) was added to the substrate. After the cultures, the yeast fats were analysed in the same manner as in example 1.

The baking yeast had a fat concentration of 18.2 w% of the yeast dry substance when the substrate contained alpha linolenic acid and in the presence of 15.9 w% of gamma linolenic acid (figure 3). Triglycerides accounted for 70 w% of the cell fats (98 mg/g of dry substance) in the presence of alpha linolenic acid and 65 w% (88 mg/g of dry substance) in the presence of gamma linolenic acid. The amount of polar lipids did not change notably (34-40 mg/g). Figure 4 illustrates the relative proportions of different fatty acids in triglycerides and polar lipids. The esterification degrees of alpha and gamma linolenic acids in the total fats of the cells were 57% and 53%, respectively. The concentrations of monounsaturated fatty acids decreased at the expense of these added acids. The test indicates that yeast can be used also for the esterification of acids of the omega-3 series and the omega-6 series.

Example 3

The possibility of replacing baking yeast with other yeasts was tested by cultivating the yeast Kluyveromyces marxianυs (Candida kefyr) under the conditions of example 1 in a substrate containing 0.2 mg/ml of CLA (Sigma).

The yeast Kluyveromyces marxianus ATCC 42265 was cultivated in 50 ml of YM broth (Difco). The substrate had pH 6.2. The cultivation period was 17 h at a temperature of 25 ⁰C and during agitation (250 rpm). 4% by volume of this culture was inoculated in an identical culture broth, to which 0.2 mg/ml of CLA had been added. The cells were cultivated for 17 h as above. Cell separation and analysis of the fatty acid composition were performed as in example 1.

A comparison of the fat of cells grown in the presence of CLA to the fat of the control cells showed that CLA replaced almost completely the linolenic acid and palmitoleic acid in the fats of this particular yeast (figure 5). Of all of the fatty acids of the corresponding lipid class, 65 w% of CLA had been esterified to the polar lipids and 67 w% to the triglycerides, so that the overall proportion of esterified CLA accounted for 47 w% of all of the yeast fats (figure 6).

Example 4

Esterification of free CLA to the fats of baking yeast was performed by cultivating yeast in a 5 w% mixture of oat and water, in which the CLA had been produced by microbiological means. The CLA production was obtained by utilising the lipase activity of oat, which hydrolyses oat fat into free fatty acids and by adding propionic acid bacteria to the mixture, which, in turn perform isomerisation of the released linolenic acid into CLA. The control comprised a mixture of oat and water containing CLA, which had been treated in a corresponding manner and to which no baking yeast had been added.

The oat grains were ground and mixed in water so as to yield a 5 w% mixture of oat and water. Hydrolysis of the fat in the oat mixture and isomerisation of released linolenic acid to CLA by means of propionic acid bacteria cells were performed as in the reference Vahvaselka, M., Lehtinen, P., Sippola, S., Laakso, S., 2004, Enrichment of conjugated linolenic acid in oats (Avena sativa L.) by microbial isomerization, J. Agric. Food Chem. 52, 1749-1752. The hydrolysis step of the oat fat was performed without pH adjustment at a temperature of 28 ⁰C and its duration was 17 hours. After this, the isomerisation reaction was activated by raising the pH of the mixture to 8.5 and by pH adjustment whenever necessary. The isomerisation step had a duration of 25 hours. Then the pH of the mixture was adjusted to 7.5 and the mixture was heated at a temperature of 90 ⁰C for 10 min. 2 volume per cent of yeast cell inoculum Saccharomyces cerevisiae (strain VTT- 72021 ), which had been cultivated in wort broth as in example 1 , was added to the cooled mixture (50 ml). The yeast was cultivated in an oat mixture containing CLA for 24 hours at a temperature of 30 ⁰C, agitation 270 rpm. The control comprised a mixture of CLA and oat, to which no yeast cells had been added. The fatty acid composition of the lipid classes was determined on the solid matter of the mixtures as in example 1.

After isomerisation performed by the propionic acid bacteria cells, the CLA content of the oat mixture was 0.35 mg/ml (8.4 mg/g of dry substance). When baking yeast had been cultivated in the mixture for 24 hours, the proportion of CLA esterified to triglycerides had increased to 33 w%. The results prove that microbiological esterification of CLA can be carried out in connection with microbiological isomerisation.

Claims

1. A process for modifying a fatty acid ester composition in an edible product, with at least a portion of the fatty acid esters converted to a fatty acid ester, whose fatty acid residue contains a fatty acid residue having beneficial health effects, characterised in comprising the following steps

- a free fatty acid having beneficial health effects is added to or produced in a mixture containing nutrients necessary for an eucaryotic micro-organism, - an eucaryotic micro-organism is inoculated in the mixture containing fatty acid,

- the eucaryotic micro-organism is cultivated in order to obtain a microbial mass in the substrate, so that the free fatty acid having beneficial health effects migrates to the cell and is esterified to fatty acid ester under the action of the eucaryotic micro¬ organism and - the microbial mass containing fatty acid esters thus obtained is added to an edible product or is used as an edible product.

2. A process as defined in claim 1 , characterised in that at least part of the fatty acid esters is a triglyceride, in which 1-3 of the acyl groups is a residue of said fatty acid having beneficial health effects.

3. A process as defined in claim 1 or 2, characterised in that the fatty acid having beneficial health effects is a conjugated linolenic acid, an omega-3 or omega-6 fatty acid or a mixture of these.

4. A process as defined in claim 3, characterised in that the fatty acid having beneficial health effects comprises the conjugated linolenic acid.

5. A process as defined in claim 4, characterised in that the conjugated linolenic acid has been obtained by isomerising linolenic acid derived from corn.

6. A process as defined in any of claims 1-5, characterised in that the mixture used for micro-organism culture contains corn.

7. A process as defined in any of claims 1-6, characterised in that the eucaryotic micro-organism is yeast.

8. A process as defined in claim 7, characterised in that the yeast pertains to the genii Saccharomyces, Kluyveromyces or Candida.

9. A process as defined in claim 8, characterised in that the yeast is Saccharomyces cerevisiae or Kluyveromyces marxianus.

10. A process as defined in any of claims 1-9, characterised in that under the esterification conditions the eucaryotic micro-organism is unable of isomerising the fatty acid into another fatty acid.

11. A process as defined in any of claims 1-10, characterised in that the microbial mass and the substrate are added to an edible product or is used as an edible product.

12. A process as defined in any of claims 1-10, characterised in that the microbial mass or alternatively the microbial mass and the substrate are dried and added to an edible product or is used as an edible product.

13. An edible product, characterised in containing an eucaryotic micro- organism, the esterified fatty acids in the cells or cell parts of the eucaryotic micro¬ organism having a composition such that the proportion of the fatty acid residue having beneficial health effects has been increased or a fatty acid residue having beneficial health effects has been formed therein.

14. An edible product as defined in claim 13, characterised in that at least part of the esterified fatty acids is a triglyceride, with 1-3 of its acyl groups being a residue of said fatty acid having beneficial health effects.

15. An edible product as defined in claim 13 or 14, characterised in that the fatty acid having beneficial health effects is a conjugated linolenic acid, omega-

3 or omega-6 fatty acid or a mixture of these.

16. An edible product as defined in claim 15, characterised in that the fatty acid having beneficial health effects comprises the conjugated linolenic acid.

17. An edible product as defined in any of claims 13-16, characterised in that the eucaryotic micro-organism is yeast.

18. An edible product as defined in any of claims 13-17, characterised in that it can be prepared by a process as defined in any of claims 1-12.