WO1999007820A2

WO1999007820A2 - Lipid removal

Info

Publication number: WO1999007820A2
Application number: PCT/GB1998/002332
Authority: WO
Inventors: Robert Muller; Kamini Dickie; Charles Bamforth
Original assignee: Brf International
Priority date: 1997-08-05
Filing date: 1998-08-04
Publication date: 1999-02-18
Also published as: GB9716578D0; AU8637598A; WO1999007820A3

Abstract

A method for removing lipids material from beer or other beverages. The beer is contacted with immobilised lipid binding protein which binds any lipid present in the beer or other beverage. After the lipid removal stage is complete there are no lipid binding additives remaining in the beverage.

Description

LIPID REMOVAL

The present invention relates to a method and apparatus for removing lipid material from solution and in particular, the present invention relates to a method and apparatus for removing lipid material from beer or other beverages.

The presence of lipid in beer can influence it's quality in a number of ways. Most importantly, lipids can cause foam damage and can affect the flavour stability of the beer.

A head of foam on a glass of beer is one of the most important quality criteria for the final product. It is one of the first features noticed by the customer and thus has a major influence on the "first impression" created by the product in the mind of the consumer.

Beer foam is composed of many interacting beer components including protein, hop materials and divalent cations. (Slack, P.T. , and Bamforth, C.W. (1983) J.Inst. Brew. 89,397-401 ; Yokoi, S. , et al, (1994) J. Am. Soc. Brew. Chem. 52, 123-126; Simpson, W.J. , and Hughes, P.S. , (1994) Cerevisia Biotrchnol. 19,39-44.

Lipids have a very detrimental effect on beer foam quality. Lipids such as those found in crisps or lipstick or those derived from yeast or malt cell walls can interact with foam proteins thereby destabilising the complex. The mechanism for this has not been clearly identified but, because the hydrophobic nature of beer proteins is important for their ability to promote foaming, it is likely that lipids interfere with this interaction. It has been noted that beer that contains lipid material shows poor foam stability. (Haboucha, J., et al, (1981) Proc. Eur. Brew. Conv. Copenhagen O,

451-459; Roberts, R.T. , et al (1978) J. Inst. Brew. 84, 9-12).

Some proteins, when added to beer are able to counteract the effects of lipids. Examples of such a protein are lipid binding protein (LBP) which is found in wheat and barley. Addition of partially purified LBP to beer can aid recovery of a beer foam that has been challenged with lipid material. Again, the mechanism by which

this works has not been fully identified but it is likely that LBP is able to bind lipids in such a way that they no longer inhibit the foam enhancing properties of the beer proteins present. (Clark, D.C. , et al, (1994) J. Inst. Brew. 100,23-25).

Flavour deterioration in beer probably proceeds by several different mechanisms but one that has been clearly identified is the formation of trans-2-nonenal from unsaturated fatty acids. Trans-2-nonenal imparts a "cardboard" flavour to beer and can produce a musty aroma. It is found in stale beers at a level above its flavour threshold of 0.1 1 ppb. This compound is derived from unsaturated fatty acids, either by direct autooxidation or through the effects of lipoxygenase (Bamforth, C.W. (1986) The Brewer 72,48-51; Drost, B.W., et al, (1990) J. Am. Soc. Brew. Chem. 48, 124-131 ; Tressl, R. , et al, (1979) Proc. Eur. Brew. Conv. Berlin 0, 27-41 and Meilgaard, M.C. (1975) Tech. Q. Master Brew. Assoc. AM. 12, 151-168. Although it is generally assumed that lipids cause problems in brewing, they do also have some beneficial effects. This is clearly demonstrated by the beneficial effects of lipids on yeast growth during fermentation. Although fermentation is necessarily anaerobic, in fact, yeasts do require a small amount of oxygen at the early stages of fermentation. The purpose of this is to synthesise the unsaturated fatty acids required for cell wall formation. Small quantities of the fatty acids can substitute for oxygen in experimental systems indicating the possible benefit of these during fermentation. (Andreasen, A. A. , and Stier, T.J.B. (1953) J. Cell. Comp. Physiol.

41, 23-36; David, M.H. , and Kirsop, B.H. (1973) J. Inst. Brew. 79, 20-25;

Thurston, P. A. , et al, (1982) J. Inst. Brew. 88, 90-94.

The types of lipids shown to be present in worts include triglycerides, phospholipids, and steryl esters but are predominantly free fatty acids. Of these, only the triglycerides would normally pass through the fermentation process as the others would be consumed by the yeast. Of 277g of total lipid in 8800g of malt,

only 0.03% remains in the finished beer (Anness, B.J., and Reed, R.I.R., (1985a) J. Inst. Brew. 91 , 312-317; Anness, B.J. , and Reed, R.J.R. (1985b) J. Inst. Brew. 91, 82-87.

The principal sources of lipids in beer are two-fold. They may originate in the brewing process itself, for example, they may be derived from barley malt or from a brewing adjunct such as a maize product. Alternatively, yeast synthesise lipids during the fermentation process. Although these should not leak out of the yeast, it is possible, and under some circumstances likely, that this happens. Lipids may also be introduced into the product at the point of consumption. In both cases, it is desirable to be able to monitor and quantify the lipid material causing any problem.

It has been previously proposed to stabilise beer foam against lipid damage by adding propylene glycol alginate (PGA) to the beer. Part of the mechanism by which this works is believed to be the ability of PGA to bind lipid. PGA is added to the beer as an ingredient and is not removed prior to consumption of the end product (Bennett, A, N. (1993) Inst. Brew (Centr and Sth Africa Sec) 4, 185-193).

It has also been proposed to add lipid binding protein to beers in order to improve foam stability. The LBP is included as an additive and is not removed from the beer after its action is complete. It therefore remains in the beer for consumption. (Kunst, A. , et al. (1997) Pat. WP1 97 - 145708/13).

It has been previously proposed to quantify lipid content in beer in a number of different ways:-

Lipids in beer may be determined using solvent extraction followed by analysis using high performance liquid chromatography (HPLC). Although this method is sensitive and has a suitably low detection limit, it is time consuming and requires sophisticated equipment with trained operators (Morrison, W.R. , et al, (1980) J. Sci. Food Agric. 31 , 329-340, Tweeten, T.N., and Wetzel, D.L. (1983) Cereal Chem. 60, 411-413).

Lipids may also be determined using enzyme based systems such as that available in kit format from Sigma Chemical Company. The detection limit with this system, however, is generally not low enough to identify lipids at the levels that are able to

damage beer quality.

It has also been proposed to use a column based system to remove lipids from beer. This system uses a column consisting of a non-specific cellulose matrix (Preptube) ( ackson, G. , 1981) J. Inst. Brew. 87,242-243). This system is able to remove lipids from beers when the lipid has been added as part of an experimental protocol. There is no evidence, however, that the cellulose matrix can remove lipid that is already bound to protein in the beer. Indeed, it is shown that protein itself is not removed by the column. Accordingly, such a column based system is not able to remove lipids present naturally in beer and thus cannot be used to improve the foam stability of commercial beers. Moreover, this column based system is not able to detect lipids derived from the brewing process in beers even when a high lipid content has

been identified by other methods.

It is an object of the present invention to provide a method and apparatus for determining lipid content in beers or other beverages.

It is another object of the present invention to provide a test for beer foam and flavour quality.

It is another object of the present invention to provide a method and apparatus for stabilising foam against lipid damage by removing lipid from the beer.

It is a further object of the present invention to provide a method and apparatus for removing lipids materials from beer or other beverages during manufacture.

According to a first aspect of the invention, there is provided a method for removing lipids material from beer or other beverages characterised by contacting the beer or other beverages with an immobilised protein said protein being capable of binding and retaining lipids material in the beer or other beverage.

According to a second aspect of the present invention, there is provided apparatus for removing lipid materials from beer or other beverages said apparatus comprising a vessel containing an immobilised protein, said immobilised protein being arranged such that fluid can pass over it and wherein the immobilised protein is capable of binding and retaining lipid materials from the fluid.

In ^"the present invention, the term "lipid binding protein" is used generically to define any protein capable of binding lipids. It is envisaged that different lipid binding proteins may be used to achieve different aims with regard to e.g. binding efficiency, cost or food acceptability. Any protein able to bind lipid could be used, however, preferred examples include lipid binding protein (LBP) from wheat, lipid transfer protein (LTP) from wheat, hydrolysed egg albumin or bovine serum albumin.

A skilled person will be aware of methods of immobilising proteins and examples of base matrices that could be used as supports for entraping or attaching the lipid binding protein. Many different types of matrix are available and the choice of

immobilisation method may depend on the particular lipid binding protein used and whether the method is to be used for analytical purposes or production purposes.

According to the method and apparatus of the present invention lipids bind to the lipid binding protein immobilised in or on the matrix/support and are thus removed from the beer or other beverage. The protein used to bind the lipid does not remain

in the beer or other beverage after its action is complete. According to the present invention there are no lipid binding additives remaining in the beer or other beverage after the lipid removal stage is complete.

In order to achieve the first object of the invention, to determine lipid content in a beer or other beverage, an insoluble support bearing the immobilised lipid binding protein may be arranged in a column. The beer or other beverage may be applied to the top of the column. As the fluid passes through the column any lipids materials present in the beer are bound to and retained by the immobilised protein. The column may then be washed with an appropriate buffer such as phosphate buffered saline or acetic/acetate buffer to remove any residual beer or other beverage whilst retaining the protein bound lipid. After all other material has been cleaned from the column, the lipids can be eluted using an appropriate solvent or an aqueous buffer

treatment. Examples of appropriate solvents include hexane, methanol, acetonitryl or combinations thereof. The lipid quantity can then be determined by previously proposed techniques such as HPLC, GC or colorimetric assay. Alternatively the change in lipids content in the beverage can be determined by subtraction using

HPLC, gas chromatography (GC) or colorimetric assay. In such a method the

column step concentrates and partially purifies the sample to be quantified and can be used to detect and quantify lipid content in the beer or other beverage.

The overall quality of the beer or other beverage can be assessed by analysing the influence of lipid content on foam quality and/or flavour quality.

The effect of lipid content on foam quality may be quantified by a method comprising the following steps: Determining the foam quality of a beer or other beverage. A skilled person will be aware of known methods for assessing foam quality and examples include measuring the rudin head retention value (Rudin, A.D.J. Inst. Brew., 63, 1957, 506-509) or the NIBEM (Klopper, W.J., Brewers Digest, 52, 1977: 51-52) method. The beverage sample is then passed through a column containing immobilised lipid binding protein. The foam quality of the

beverage sample is then re-assessed using the same method as the first stage. The final foam quality (or foam performance) can then be compared to the initial foam quality (or foam performance). Any difference is indicative of the presence of foam negative materials (lipids). If the test indicates the presence of foam damaging lipids, remedial steps can be taken to ameliorate the problem.

The effect or influence of lipid content on flavour quality of a beer or other beverage can be assessed by the following method:

Firstly the flavour quality of the beer or other beverage is assessed. Methods for assessing this characteristic will be known in the art and examples include organoleptic analysis (Meilgaard, M.C. Tech. Q. Master Brew. Ass. AM. 1975, 12, 151-168) ageing, forced ageing or assessment of carbonyl content (Devreux, A. et

al - Proceedings of the EBC Congress, 1981 : 191-201). The beverage is then passed through a column containing immobilised lipid binding protein. After passage through the column, the flavour quality is re-assessed using the same protocol as the first stage. The final flavour quality measurement can then be compared with the initial flavour quality measurement. Any difference is indicative of the presence of flavour damaging lipids in the beverage and remedial steps may be taken during the manufacturing process to ameliorate the problem.

In ^"order to achieve the further object of removing lipids materials from beer or other beverage during manufacture, an insoluble matrix support bearing immobilised lipid binding protein may be arranged in a column or bioreactor type device. Typically this column or bio-reactor device may be on a larger scale than that proposed for use in quantifying lipid content. The column or bio-reactor device may be arranged in-line in the brewing production line such that the beer or other beverage can be applied to the column or bio-reactor type device in a continuous flow. As the fluid passes through the column, any lipid materials present are bound to and retained by the immobilised lipid binding protein. After a defined residence time passing through the column or bio-reactor type device, the lipid depleted beer or other beverage may pass onto the next stage of the production process. At intervals, the column can be replaced or taken off-line for regeneration by elution of the bound lipid using an appropriate solvent or buffer system.

Typically the insoluble matrix bearing the immobilised lipid binding protein will be arranged in a column or bio-reactor type vessel however it is also envisaged that immobilised lipid binding proteins could be used as a processing aid in the brewing process. For example, lipid binding proteins could be immobilised onto beads or pellets for adding to the beverage. Lipid material could be removed from a beverage by introducing a pre-determined quantity or charge of immobilised lipid binding proteins as an additive to the beverage during the production process and allowing it to remain in contact with the liquid for a pre-determined residence time. After the defined period, the immobilised lipid binding protein can be removed by a known separation method such as filtration, centrifugation or settling.

The residence times required to remove lipids materials from the beverage will depend on factors such as whether the process is analytically or production based, the lipid binding protein used, the type of immobilisation used and the support or matrix type. The appropriate residence time could be determined empirically by a skilled person.

It is also envisaged that immobilised lipid binding proteins can be regenerated after use. After treatment of the beer or other beverage, the lipid binding sites of the lipid binding protein may be more or less saturated with lipid thereby reducing subsequent binding capacity. This binding capacity can be regenerated using a cycle of washes at different pH. The actual cycle used will depend on subsequent applications but a typical example could comprise the following steps:

1) pass the beer or other beverage through the column;

2) wash the column with 0.1 M acetate pH 2, 0.5 M NaCl;

3) wash the column with 0.5 M ethanolamine pH8.3, 0.5 M NaCl;

4) repeat step (2) from 0-n times;

5) repeat step (3) from 0-n times;

6) wash the 0.1 M acetate pH 4, 0.5 M NaCl.

The immobilised lipid binding protein is then ready for further use.

Reference is now made to be following illustrative examples.

Figure 1 is a schematic representation showing removal of lipid by an insoluble support having protein bound thereto; Figure 2 shows foam stability of samples of degassed beer in the presence and absence of LBP and/or lipid. The figure shows the effect of lipid binding protein added to lipid challenged beers. Alone (100 μ g/ml) lipid binding protein was not able to improve the stability of this standard beer. When the beers were treated with 2μg/ml of either linoleic acid or phospholipid the foam stability fell dramatically. The addition of 100 μg/ml of lipid binding protein was able to restore the foam stability of the lipid - challenged beers; and

Figure 3 shows foam stability of commercial beer samples in the absence and presence of LBP. The¹ figure shows that whereas the lipid binding protein itself does not improve foam stability in commercial beers with good foamability, it is able to improve some beers. This is attributed to the ability of lipid binding proteins to bind lipid specifically and avidly. That many beers were not improved implies that they did not suffer from lipid problems. That some beers were improved implies these did suffer from lipid problems.

EXAMPLE 1

A method for analysing the influence of lipid on beverage foam quality.

Sample beers were obtained from a local market without any prior knowledge of the

provenance of those beers.

Samples were tested for foaming quality by one of three methods:

(A) Visual appearance

(B) Rudin Head Retention Value (measuring liquid drainage from the foam J. Inst. Brew, 63, 1957: 506-509) (C) NIBEM (measuring foam stability Klopper, W.J., Brewers Digest,

52, 1977:51-52). Each beer was passed down a column containing immobilised LBP and the foam measurement repeated.

(A) The visual appearance of the foam was reported as improved on some beers

after column treatment. In Figure 4 the control beer is on the left the treated beer is on the right. The volume of foam produced (by a standard procedure) is clearly increased in the treated beer.

(B) Figure 5 shows that the head retention value of some commercial beers was improved (increased) after column treatment. This implies that some beers contained foam suppressing agents that were removed by the column. Foam improvement in some beers was greater than in others indicating a low level of foam negative agents in the latter.

(C) Figure 6 shows the NIBEM values of a beer before and after treatment.

Treatment of the beer has resulted in more foam stability at each of the recording times again indicating an improvement in quality.

Thus it was possible to show that some commercial beers do not foam as well as they potentially could and that using immobilised LBP achieved improved foamability.

EXAMPLE 2 A method for analysing the influence of lipid on flavour quality of a beverage.

The aromas profile of a beer was described by a trained beer flavour panel. The same beer was then treated by passage through an immobilised LBP column. The beer was profiled again by the same panel. Treatment with the column was shown to remove certain aromas and improve organoleptic quality. Table 1 details some of these improvements.

Thus it was possible to show that LBP treatment of lipid damaged beer could improve organoleptic quality.

EXAMPLE 3

A method for analysing the lipid content of a beverage.

(A) Following polar solvent extraction and derivitisation, the lipid materials of a beer were analysed by GC.

(B) Further sample of the same beer was treated by immobilised LBP and the

procedure above was repeated on the eluant.

Table 2 shows that the level of several fatty acids were reduced.

Thus it was possible to exploit the LBP column system to aid in the identification of foam damaging lipids.

EXAMPLE 4 A method of removin lipid material from a beverage: Process aid

In this example silica hydrogel was treated with lipid binding protein in-order to prepare an immobilised lipid additive that could be removed from the beer by physical means. Figure 7 shows that:

Addition of silica hydrogel gel or protein treated silica hydrogel gel did not have a significant effect to the foam quality (head retention value) of the beer.

The addition of lipid to the beer, with or without silica hydrogel, resulted in a dramatic reduction in foam quality.

The addition of lipid binding protein silica hydrogel to lipid damaged beer with subsequent removal of the lipid/lipid binding protein/silica hydrogel resulted in restoration of the foam quality.

Thus the quality of beer could be improved by the addition and subsequent removal of an immobilised lipid binding protein.

EXAMPLE 5

A method of removing lipid material from a beverage: Bioreactor

Figure 8 shows a time course of lipid removal from beer during sequential sampling. The flow rate was lml/min.

(A) An addition of 2mg/ml of linoleic acid was added to beer in-order to mimic lipid damaged beer. (B) The beer was pumped through a column of immobilised LBP in a continuous manner.

(C) The head retention value of the eluant beer was determined and is shown as a function of time in Figure 8.

Thus it was possible to improve beer quality by continuous passage of the beer through a bio-reactor type system.

EXAMPLE 6

Regeneration of the Lipid Binding Protein after Use.

Figure 9 shows the effect of an acid/base wash of the column following previous usage. The regime used was as follows (also previously described): i) Use column ii) Wash column with 0.1 M acetate pH 2, 0.5 M NaCl. iii) Wash column with 0.5 M ethanolamine pH 8.3, 0.5 M NaCl. iv) Repeat ii) v) Repeat iii) vi) Wash with 0.1 M acetate pH 4, 0.5 M NaCl in preparation for further use.

The samples examined were: a) The control beer. b) The control beer after LBP treatment - showing an improvement in foam quality. c) The control beer after treatment on a column that had been saturated with

lipid, showing no improvement. d) The control beer after treatment on a regenerated column once again showing improvement.

This demonstrates that a cycle of washes at appropriate pH can regenerate the lipid binding capability of the immobilised LBP.

The addition of bovine serum albumin (BSA) to beer could result in improved foam quality merely because BSA is itself able to enhance foam production. Thus improved foam quality in lipid damaged beers could be due to leaching of LBP from the immobilised state into solution. The following experiment demonstrates that the level of BSA required to produce the observed enhancements was much higher than the level that could be detected in the beer. Thus the improvements could not be attributed to leached BSA in the beer.

1) Level of BSA required to achieve improvement in foam.

Typically the improvement in foam quality observed was some 15-20% of the control value in the beers for which improvement was observed. It was determined, by trial and error, that an equivalent improvement could be achieved by the addition of 50 μg/ml BSA. 2) Level of BSA identified by ELISA.

The true quantity of BSA in the beers was determined by enzyme linked immunosorbant assay using an antibody specific to bovine albumin. Figure 10 shows the detection limit of BSA in beer by this method. The concentration of BSA in beers after column treatment was below the detection limit for this system, i.e. below 0.5 μg/ml. Thus the improvement in foam quality of treated beers could not be attributed to the presence of LBP in the product.

Claims

CLAIMS:

1. A method for removing lipids material from beer or other beverages characterised by contacting the beer or other beverage with an immobilised protein said protein being capable of binding and retaining lipids material present in the beer or other beverage.

2. A method according to Claim 1 for use in determining lipid content in a beer or other beverage.

3. A method according to Claim 1 for use in removing lipids materials from beer or other beverages during manufacture.

4. A method according to Claim 3 wherein the beer or other beverage contacts

the immobilised lipid binding protein in a continuous flow operation.

5. A method according to Claim 3 wherein the beer or other beverage contacts the immobilised lipid binding protein in a batch operation.

6. A method according to Claims 4 or 5 wherein the immobilised lipid binding protein is arranged in a column.

7. A method according to Claims 4 or 5 wherein the immobilised lipid binding protein is arranged in a bio-reactor.

8. A method according to Claim 3, 4 or 5 wherein the beer or other beverage is contacted with a charge of immobilised lipid binding protein.

9. A method for eluting bound lipid and regenerating lipid binding sites on immoblised protein wherein the immoblished protein is washed with a series of aqueous buffer solutions said aqueous buffer solutions being alternative low salt concentration and low pH and high salt concentration and high pH.