WO1997005272A1

WO1997005272A1 - Glycoprotein from yeast

Info

Publication number: WO1997005272A1
Application number: PCT/EP1996/002588
Authority: WO
Inventors: Anthonie Kunst; Eoin Lalor; Diederik J.-M. Schmedding; Bartholomeus Josef Van Schie
Original assignee: Quest International B.V.
Priority date: 1995-07-25
Filing date: 1996-06-14
Publication date: 1997-02-13
Also published as: AU6303796A; ZA965374B

Abstract

The invention provides a glycoprotein obtainable by incubating a food grade yeast material suspended in an aqueous medium with an enzyme preparation comprising β-glucanase activity at an initial pH from 3.0 to 5.5, followed by heat treatment of the liquid obtained at an initial pH between 5.5 and 9.0 and optionally followed by separating solid material and/or other conventional aftertreatment. Preferably the yeast material is an intact food grade yeast. Preferably the incubation is carried out at 30-60 °C for 30 minutes to 30 hours, followed by heat treatment above 70 °C for a period of 2 seconds to 60 minutes. The glycoprotein is useful as a foam stabiliser for foodstuffs and beverages, especially beer.

Description

Glycoprotein from yeast

The invention relates to a glycoprotein obtainable from yeast. More in particular the invention relates to a glycoprotein obtainable by digestion of a yeast material, such as e.g. Saccharomyces cerevisiae, with certain enzyme preparations.

Glycoproteins have certain surface active properties and such products derived from natural sources may have cer¬ tain advantages over chemically synthesized surface active agents. A specific class of glycoproteins viz. mannopro- tein from certain yeasts have been investigated in some depth and the results reported: David R Cameron et al disclose in Applied and Environmen¬ tal Microbiology, June 1988, 1420-1425 mannoprotein emul¬ sifiers e.g. obtained from Saccharomyces cerevisiae. Two preparative methods were used viz autoclaving in neutral citrate buffer and by digestion with Zymol(y)ase 20T, ex Miles Laboratories, Toronto, Canada, containing a β-1,3- glucanase for 3 hours at 25°C and pH 7.5 and an emulsifier was precipitated. Many yeast species were tested, includ¬ ing 13 genera other than Saccharomyces. The ratio of mannose to protein of the purified emulsifier obtained from S. cerevisiae was stated to be 44:17 or 2.6:1. Atten¬ tion in this publication was focused on using spent yeast from the manufacture of beer and wine as a possible source for the large scale production of mannoprotein bioemulsi- fier. In 1993 David R Cameron disclosed in a dissertation from McGill University; Can. : "Identification and characteriza¬ tion of mannoprotein emulsifier from bakers' yeast" (ace. to Diss. Abstr. Int. B 1994, 55(6), 2076) further work on the mannoprotein emulsifier fractionating the material into higher and lower molecular weight fractions with different physico-chemical properties. The mannoproteins were obtained by autoclaving yeast cells in a neutral buffer (evidently in the absence of added enzyme and it was stated that spent yeast from the beer and wine indus- tries is suitable raw material. These products were not found quite effective for certain applications and there is room for considerable improvement.

In a first embodiment the present invention provides a glycoprotein obtainable by incubating a food grade yeast material suspended in an aqueous medium with an enzyme preparation comprising β-glucanase activity at an initial pH from 3.0 to 5.5, followed by heat treatment of the liquid obtained at an initial pH between 5.5 and 9.0 and optionally followed by separating solid material and/or other conventional aftertreatment of the liquid.

Under food grade yeast is here to be understood such a yeast of the genera Saccharomyces, Kluyveromyces, Candida and Torula and any such microorganisms with genetic material thereof coding the structure or regulator genes of the glycoprotein/cell wall material. More preferably an intact food grade yeast is used, in particular of the genera Saccharomyces and Kluyveromyces and Saccharomyces genera such as Saccharomyces cerevisiae strains are par¬ ticularly preferred. It is also possible to use a yeast material e.g. a deactivated yeast, a yeast with (mech¬ anically) broken cell walls, fragments or certain frac¬ tions of yeast etc. Spent yeasts e.g. from wine making may be acceptable, but partially autolysed yeasts are some¬ times less suitable because of certain chemical changes which may have taken place.

The β-glucanase containing enzyme preparation can be obtained from various sources such as plants or microor- ganisms. β-1, 3-glucanase is preferred. The preparation can be a crude cell material or rather a purified material. The use of purified enzyme materials low in protease activity is preferred. Suitable commercially available sources are: Bioglucanase EL, ex Quest International, Naarden, Netherlands; Viscoenzyme, ex Novo Nordisk A/S, Bagsvaerd, Denmark and Zymolyase, ex Miles Laboratories, Toronto, Canada, the latter especially if the protease component is deactivated. Enzyme preparations having both β-1,3-glucanase and cellulase activity are sometimes pre- ferred. The amount of enzyme preparation used is usually 0.1 to 100 g Bioglucanase EL per gram of yeast material as dry matter, more preferably 1 to 50 g Bioglucanase EL are used per gram of yeast material. Bioglucanase EL contains 900 BG units per gram of Bioglucanase liquid. BG units are defined in Standard Analytical Methods SAM 0109-1 avail¬ able from Quest International, Naarden, Netherlands. In the case of other enzymes being used other relative quan¬ tities may be used depending on the activity of the enzyme. The expert knows how to calculate the relative amount of enzyme to be used based on the activity of the enzyme.

The incubation with the glucanase containing enzyme pre¬ paration is preferably carried out at 30-60°C, preferably 40-55°C; an initial pH from 3.5 to 5.3, preferably 3.7- 5.0 and for 30 minutes to 30 hours, preferably 4 to 15 hours followed by heat treatment above 70°C, preferably above 80°C at an initial pH between 6.0 and 8.0, more preferably between 6.3 and 7.5 for a period of 2 seconds to 60 minutes, preferably 10 seconds to 30 minutes. In order to stabilise the pH it may be useful to include in the suspension a buffer composition as e.g. an acetate buffer with a pH of 4.5-5.0. Exact conditions applied during incubation usually also depend on activity and optimum conditions of the specific glucanase enzyme employed. The incubation is carried out in an aqueous medium meaning that the medium comprises for the greater part water, some other (organic) solvent, which does not interfere with the enzyme activity may be present. The subsequent heat treatment of the liquid obtained can be carried out at any temperature above about 70°C, pre¬ ferably above 80°C, especially above 90°C and serves to pasteurise/sterilise the liquid and also to improve the properties thereof. Heat treatment at higher temperatures i.e. above 90°C is preferred because it usually improves the quality of the glycoprotein obtained. The time of the heat treatment can vary widely, if high temperatures say 140°C under pressure is used a few seconds (UHT) may be used, but if lower temperatures are used longer heating to up to 60 minutes may be necessary. Typically 85-95°C for 1 to 30 minutes is used. Heating is conveniently effected by steam injection. It is recommendable to ensure that the initial pH of the heat treatment is between pH 5.5 and 9.0, for better results between 6.0 and 8.0, especially between 6.3 and 7.5 so that a pH adjustment is generally required e.g. by adding some alkali.

Separation of solid material can conveniently be carried out by centrifugation or filtration. Sometimes this step is preceded or followed by decolourising e.g. with absorb¬ ent like activated carbon. Another optional aftertreatment is concentrating or drying the glycoprotein solution obtained. This can be carried out by heating, often under reduced pressure, e.g. heating to about 40-75°C at a pressure of 10-100 bar for several hours such as 2-6 hours and for longer periods when drying such as for 6-10 hours. The glycoprotein obtainable by the process according to the present invention preferably has a carbohydrate:protein ratio between 1:1 to 1:10, prefer¬ able 1:3-7. With certain assumptions an estimate of the average molecular weight can be made from FPLC data. The FPLC method followed uses a Superose 12 (ex Pharmacia) column with a diameter of 1 cm and uses a 50 mM acetate buffer of pH 5 as the eluent. The elution rate was 0.7 ml/min and the elution time of the glycoprotein found is between 13 and 17 minutes. This may suggest an average molecular weight of about 150 kD. The term glycoprotein should understood to be in this specification a glycoprotein with surface activity and does not mean to imply that the product is applicable only in emulsions. In fact the surface activity especially permits other valu- able applications such as stabilisation of foams in e.g. foods and beverages e.g. foam stabilisation of protein or hydrolysate containing foams such as in beer, low alcohol beer and shandy. As a foam stabiliser in beverages the present natural glycoprotein surface active agent offers advantages over e.g. commercially available propylene glycol modified alginate which is disclosed in J. Inst. Brew. ££, 34-37 (1980) . More in particular the present glycoprotein may not only result in a better foam stabil¬ ity, it may also visually result in smaller bubbles, better resistance to negative foam factors such as e.g. lipid tolerance, and/or it may improve beer processing, may improve taste, and may prevent lipid related off-fla¬ vours etc. Generally the glycoprotein is obtainable by enzyme treat- ing the yeast material followed by heat treatment, separ¬ ating the solid material (e.g. the pellet) , collecting and heat treating the liquid optionally followed by after¬ treatment . It is also possible and sometimes advantageous to treat the yeast material with enzyme, separating the liquid from the solids, resuspending the solids, heat treating the suspension obtained, separating the solids from the liquid optionally followed by aftertreatment of the liquid. In the latter procedure several extra steps have been inserted between the enzyme treatment and the heat treatment. Sometimes the product obtained according to the latter route is more pure than the product obtained according to the former route.

In another embodiment the present invention provides a glycoprotein obtainable by carrying out the incubation in an aqueous medium comprising from 5-25, preferably 10-20 wt. % of dry yeast matter.

According to another embodiment the present invention provides a process for preparing a glycoprotein by incu¬ bating a food grade yeast material suspended in an aqueous medium with an enzyme preparation comprising β-glucanase activity at an initial pH from 3.0 to 5.5, followed by heat treatment of the liquid obtained at an initial pH between 5.5 and 9.0 and optionally followed by separating solid material and/or other conventional aftertreatment of the liquid. In a preferred embodiment the above process is carried out by incubating an intact food grade yeast.

In a preferred embodiment the invention provides a process in which the incubation is carried out at 30-60°C, prefer¬ ably 40-55°C; an initial pH from 3.5 to 5.3, preferably 3.7 to 5.0 and for 30 minutes to 30 hours, preferably 4 to 15 hours followed by heat treatment above 70°C, preferably above 80°C at an initial pH between 6.0 and 8.0, more preferably between 6.3 and 7.5 for a period of 2 seconds to 60 minutes, preferably from 10 seconds to 30 minutes.

In another embodiment the invention provides a process in which the aqueous medium comprises from 5-25, preferably 10-20 wt % of dry yeast material.

As set out above the glycoprotein according to the present invention can be used with advantage in foods (i.a. bakery and confectionery products) or beverages, more in par- ticular to improve the foam properties thereof. The inven¬ tion therefore provides in another embodiment a food or beverage comprising a glycoprotein obtainable as described above. Use of the glycoprotein according to the invention in beer to improve the foam properties thereof is also particularly valuable. The invention also provides a pro¬ cess for improving the foaming properties of foods and beverages, especially beer, by incorporating therein an amount of glycoprotein from 0.05 ppm to 500 ppm, preferably 1-100 ppm calculated as dry matter on the weight of the food or beverage. In particular the present invention provides beverages, more in particular beer (ale, bitter, low-alcohol beer and alcohol-free beer) , cola, shandy etc comprising the glycoprotein as a foam stabiliser. Although the term foam stabiliser has been used above this does not mean to say that other properties of the products are not also improved; this may be less easily quantified but there are certain (organoleptic) indications for such improvements.

The invention will now be illustrated by the following non-limiting examples. All parts and percentages mentioned are by weight unless otherwise indicated.

EXAMPLE 1

A suspension of fresh bakers' yeast (GB 2113, ex Quest International) , containing 12% dry matter was incubated with Bioglucanase EL, ex Quest International at 10 g Bioglucanase liquid per kg yeast dry matter (YDM) during 20 hours at a temperature of 50°C. Prior to the addition of the enzyme the pH of the suspension was adjusted to pH 4.5 by adding hydrochloric acid. After 20 hours the pH was adjusted to pH 7.1 by adding aqueous NaOH and subsequently heated to 95°C and kept at that temperature for 10 minu- tes. After cooling to 60°C the mixture was separated on a Westphalia disk stack separator to remove insoluble compo¬ nents. The separator effluent was concentrated via evapor¬ ation to a concentration of 60% dry matter. The glycoprotein eluted after 15 minutes which may suggest an average molecular weight of 150 kD (estimated from FPLC as set out above) and a sugar:protein ratio of 12:40 i.e. 1:3.3.

Appliga ion Example ι

A commercially available low alcohol beer (Amstel Malt) was destabilised as to foam by the addition of 2 ppm oleic acid and various quantities of the glycoprotein concen¬ trate obtained according to Example 1 was added. The bottles were closed again thoroughly shaken and stored overnight at about 9°C. Foam stability was determined with a NIBEM foam stability tester (ex Haffmans B.V. Venlo, Netherlands) . The tests are represented in Table 1 below and focuses at the 30 mm foam collapse. The data have been expressed as relative values; the standard Amstel Malt being 100% and the Amstel Malt destabilized with 2 ppm oleic acid being 0%. In this manner the "robustness" (=beer foam stability) of the beer foam was determined. The foam obtained had a finer bubble structure than beer without the glycoprotein. There was no lipid related

(oleic acid) observed by a panel of experienced tasters.

Dosage (ppm) NIBEM- value ( % )

0 . 0 0

1 . 0 28

3 . 1 36

10 . 3 51

31 . 0 68

103 . 3 65 EXAMPLE 2

The procedure of Example 1 was repeated and the solution obtained was purified by the addition of activated carbon (5% on weight) , was stirred for 30 minutes at 50°C fol¬ lowed by filtration. The resulting solution was concen¬ trated in a Bϋchi Rotavapor EL 131 to a concentration of about 60% dry matter.

Application example 2

In this example a solution of the material made according to Example 2 was applied as described in Application Example 1. The results are tabulated in Table 2 below.

Table 2

Dosage (ppm) NIBEM-value (%) 0.0 0 1.0 36

Table 2 (continued)

3.1 45 10.3 57

31.0 68

103.3 67

Example 3 The procedure of Example 1 was repeated and the concen¬ trated solution obtained was purified by ethanol. This was effected by adding so much 96% ethanol (sg 0.8090) to the glycoprotein solution that the concentration thereof became 75% and a precipitate was formed. The solution with precipitate was left overnight at 4°C and the precipitate was obtained by centrifuging from the liquid. Ethanol remaining in the precipitate was evaporated.

Application Example 3 In this example a solution of the material made according to Example 3 was applied as described in Application Example 1. The results are tabulated in Table 3 below.

Table 3

Dosage (ppm) NIBEM-value (%)

0.0 0

0.1 11

0.2 22

0.3 27

1.0 42

2.9 56

10 70

29 78

Example 4

A suspension of fresh bakers' yeast, containing 15% dry matter is incubated with 8 g/kg YDM Bioglucanase EL, ex Quest International during 20 hours at a temperature of 50°C. Prior to the addition of the enzyme the pH of the suspension was adjusted to 4.5 with hydrochloric acid. After 20 hours the pH was adjusted to pH 7.0 using aqueous NaOH and cooled to ambient temperature (20°C) . The suspen¬ sion was centrifuged and the resulting pellet was taken up in an equal weight of water and heated for 5 minutes at

95°C. After cooling to 60°C the mixture was separated on a westphalia disk stack separator to remove insoluble compo¬ nents. The separator effluent was concentrated via evapor¬ ation to a concentration of about 60%. Application example 4

In this example a solution of the material made according to Example 4 was applied as described in Application Example 1. The results are tabulated in Table 4 below.

Table 4

Dosage (ppm) NIBEM-value (%)

0.0 0

0.1 11

1.0 22

5.0 36

7.0 39

10.0 54

30.0 70

Claims

C AIMS

1. A glycoprotein obtainable by incubating a food grade yeast material suspended in an aqueous medium with an enzyme preparation comprising β-glucanase activity at an initial pH from 3.0 to 5.5, followed by heat treatment of the liquid obtained at an initial pH between 5.5 and 9.0 and optionally followed by separating solid material and/or other conventional aftertreatment.

2. A glycoprotein according to claim 1 obtainable by incubating an intact food grade yeast.

3. A glycoprotein according to claim 1 or 2 in which the incubation is carried out at 30-60°C, preferably 40-55°C; an initial pH from 3.5 to 5.3, preferably 3.70 to 5.0 and for 30 minutes to 30 hours, preferably 4 to 15 hours followed by heat treatment above 70°C, preferably above

80°C at an initial pH between 6.0 and 8.0, more preferably between 6.3 and 7.5 for a period of 2 seconds to 60 min¬ utes, preferably 10 seconds to 30 minutes.

4. A glycoprotein according to any of the claims 1 to 3 in which the aqueous medium comprises from 5-25, prefera¬ bly 10-20 wt % of dry yeast material.

5. Process for preparing a glycoprotein by incubating an food grade yeast material suspended in an aqueous medium with an enzyme preparation comprising β-glucanase activity at an initial pH from 3.0 to 5.5 followed by heat treat¬ ing the liquid obtained at an initial pH between 5.5 and 9.0 and optionally followed by separating solid material and/or other conventional aftertreatment.

6. A process according to claim 5 in which an intact food grade yeast is incubated.

7. A process according to claim 5 or 6 in which the incu¬ bation is carried out at 30-60°C, preferably 40- 55°C, an initial pH 3.5-5.3, preferably 3.7-5.0 for 30 minutes to

30 hours, preferably 4 to 15 hours and is followed by a heat treatment above 70°C, preferably above 80°C at an initial pH between 6.0 and 8.0, more preferably between 6.3 and 7.5 for a period of 2 seconds to 60 minutes, preferably 10 seconds to 30 minutes.

8. A process according to any of the claim 5 to 7 in which the aqueous medium comprises from 5-25, preferably 10-20 wt % of dry yeast matter.

9. Food or beverage comprising a glycoprotein according to any of the claims 1 to 4.

10. Beverage with improved foam properties comprising a glycoprotein according to any of the claims 1 to 4.