AU682019B2 - Novel yeast strains - Google Patents

Description

1 -1-

AUSTRALIA

Patents Act 1990 BURNS PHILP COMPANY LIMITED

ORIGINAL

COMPLETE SPECIFICATION STANDARD PATENT Invention Title: r o Novel Yeast Strains The following statement is a full description of this invention including the best method of performing it known to us:- Y- i i NOVEL YEAST STRAINS The present invention relates to a bakers yeast Saccharomyces cerevisiae which has improved temperature tolerance and/or keeping quality.

Bakers yeast is typically produced by a fed-batch fermentation process at temperatures up to about 35 0

C.

Above this temperature both yield and baking performance are usually compromised. Unfortunately, considerable heat is generated during fermentation and the fermentors must be cooled to maintain optimum conditions for yeast growth.

Due to the large culture volumes involved in commercial fermentations (typically around 100,000 litres) cooling can represent a substantial component of overall production costs, particularly in countries which experience high summer temperatures. A strain which can tolerate even small increases in production temperature, without significant loss of baking performance, is therefore desirable.

Another important feature in a bakers yeast product 20 is its keeping quality. This is the ability of the product (at 17-34% solids) to retain its baking activity during a period of storage. Depending on the availability of bakery refrigeration, the conditions of such storage Smay range from around C to in excess of 25 0

C.

The present inventor has produced a bakers yeast Saccharomyces cerevisiae strain which is improved in respect to both temperature tolerance and storage characteristics. A sample of this yeast strain, designated YTTS, was deposited with Australian Government 30 Analytical Laboratories (AGAL) at 1 Suakin Street, Pymble, New South Wales 2073, Australia on 11 March 1993 and was accorded Accession No. N93/10504.

Accordingly, in a first aspect the present invention consists in a baker's yeast Saccharomyces cerevisiae strain, the strain being characterised in that there is less than a 12% decrease in yield of grams of wet yeast at solids per 100g total reducing sugar when the strain is brewed at 37 0 C in comparison to brewing at 30 0

C.

In a second aspect the present invention consists in a baker's yeast Saccharomyces cerevisiae strain, the strain being characterised in that the strain retains at least of its activity in plain dough at 37 0 C when brewed at 37 0

C

in comparison to when brewed at 30 0

C.

In a third aspect the present invention consists in a baker's yeast Saccharomyces cerevisiae strain, the strain being characterised in that the strain brewed at 0 C retains at least 95%, End preferably at least 97%, of its activity after storage for 21 days at 4 0 C at 24.5% solids.

The present invention also relates to baker's yeast Sacc'aromyces cerevisiae which have more than one of the characteristics specified in the first, second and third aspects of the present invention.

In a preferred embodiment of the present invention the Saccharomyces cerevisiae strain is strain YTTS 20 (AGAL N93/10504).

In a fourth aspect the present invention consists in a fresh or dry bakers yeast composition, the composition being characterised in that it includes bakers yeast of the first, second or third aspect of the present invention.

As stated above a Saccharomyces cerevisiae strain of the present invention, designated YTTS, was deposited with AGAL. In order that the nature this strain may be more .clearly understood its performance was compared with 30 another yeast, Strain A (also deposited with AGAL, Accession No. N94/8029), representative of those currently used in markets where temperature tolerance and keeping qualities are important. This comparison' is followed by a list of further characteristics of strain YTTS.

Production and Preliminary Assessment of Hybrid Yeast Strains Hybrid yeast strains were produced using classical methods of the sort described in "Yeast Genetics a manual of methods" (Spencer Spencer D.M. and Bruce Springer-Verlag, Berlin Heidelberg, 1989).

In a typical procedure strains of diploid or higher ploidy are subjected to manganese mutagenesis (Putrament Baranowska H. and Prazamo Molec. Gen.

Genet., 126: 357-366, 1973) to recover mitochondrial antibiotic resistance iutants. The strains are subsequently sporulated by incubation at 20 0 C on a solid medium comprising 0.5% w/v potassium acetate and 1% agar.

After 5-6 days asci are harvested and matable forms recovered by random spore isolation, using the ether method of Dawes and Hardie (Molec. Gen. Genet., 131: 281-289, 1974) to kill un-sporulated vegetative cells.

Spores are separated from any remaining vegetative cells by mixing the suspension with an equal volume of mineral 20 oil then allowing the spores to partition into the oil phase. They are subsequently germinated by spreading to nutrient agar plates. The mating type of the resulting haploid colonies is determined by agglutination with tester strains and their resistance to selective markers confirmed. Novel hybrids are formed by crossing haploids ecarrying complementary markers using the drop overlay met'.)d (Spencer et al., op cit, p4-5). Selection against un-mated cells is on 4% glycerol YEP plates supplemented with the appropriate antibiotics. Hybrids are identified 30 by their ability to sporulate and by the presence of both parental markers.

Baking performance of hybrids is predicted in the laboratory by an adaptation of the thin flour dough method of White (p 403, in Yeast Technology, Chapman and Hall, 1954). In this test 170mg of wet yeast (50mg dry weight) is suspended in 30ml of 0.33mM NaCl then mixed into 28g of flour for 1 minute. The 50ml thin dough is transferred to a 100ml measuring cylinder then incubated at 30 0 C. The activity of a novel hybrid relative to a control strain is determined by noting the change in volume of the doughs at 30 minute intervals. Strain YTTS was identified as having commercial potential on this basis and was further characterised by comparison with Strain A as described below.

Assessment of Strain YTTS Pilot scale yield, baking and keeping performance of strain YTTS were compared with Strain A.

Comparison 1 Method Twenty litre fed-batch fermentors were inoculated at a rate of 12% (seed:final yeast) and brewing commenced at 30 0 C. In one series of experiments this temperature was maintained throughout the brew. In a second series of experiments the temperature was gradually increased, in 2 0 C increments, from 30 0 C at hour one to reach 37 0 C by the end of hour five, where it was then maintained. In all cases a total of 1200g of total reducing sugar (TRS) was used (as 50% cane/50% beet molasses). All brews were supplemented with KC1, MgSO 4 ZnSO 4 vit B1 and vit B6 and molasses feed rates controlled to produce a similar fermentation profile for both strains.

All activity tests were carried out using an SJA Fermentograph set at 30 0 C or 37 0 C using the dough formulations shown in Table 1. Dry ingredients were weighed into a Farinograph mixing bowl and blended for one 30 minute. Compressed yeast was suspended in water then added to the dry ingredients, together with the salt solution. The complete dough was mixed for three minutes, reaching a final dough temperature of 30 0

C.

The finished dough was transferred to a standard Fermentograph tin then put into the SJA apparatus. Tests were carried out for two hours knocking the dough down after the first hour.

Baking tests were performed according to two commonly used baking processes (CDD and Sponge and Dough).

In the chemical dough development (CDD) method the ingredients listed in Table 2 were assembled in a high speed mixer and mixed for six to eight minutes. The finished dough temperature was 30 0 C. The dough was rested on the bench for 10 minutes, scaled off at 770g, molded then given a further 10 minutes rest time. After further molding, the dough piece was placed in a baking pan then transferred to a proofing cabinet set at 40 0 C and relative humidity. The time taken for the dough piece to reach a height of 120mm (the "proof time") was noted and the dough then baked for 30 minute in a rotating oven set at 220 0 C. The loaf was cooled overnight and oven spring o determined by subtracting proof height (120mm) from the final height of the loaf.

The sponge and dough method differed from the CDD 20 process in that the ingredients (Table 2) were added in two stages. The sponge stage ingredients were mixed in a high speed mixer for three minutes, achieving a final temperature of 30 0 C. The mixture was then fermented for four hours at 30 0 C in a separate vessel, knocking down after two hours. At the end of this time the sponge *4 mixture was transferred to a mixer and the remaining ingrecdients added to produce the dough stage. This was then mixed for five and a half minutes, again reaching a finished dough temperature of 30 0 C. The dough was rested 30 on the bench for 10 minutes and the CDD procedure followed for the remainder of the baking process.

Results Table 3 displays brew summaries and fermentograph activity results. The data show that the YTTS has tolerated the increase in brewing temperature (from 30 0

C

to 37 0 C) better than Strain A, with a 10.1% loss in yield in comparison with a 14.5% loss for Strain A. Dough activities at 30 0 C in all dough types were reduced for both strains as a result of brewing at 37 0 C. However the averaged results of the 37 0 C brews when compared to 30 0

C

brews, reveal that, in every dough type, the loss of activity associated with growth at higher temperature is greater with Strain A than with YTTS.

Although 30 0 C is often used for fermentograph testing, bakery proof temperatires are commonly higher than this. A second series of fermentograph tests were therefore performed, this time with the SJA apparatus set at 37 0 C. These results are also shown in Table 3. In this case, significant strain differences were noted in the losses recorded following growth at 37 0

C.

The fermentograph results are broadly reflected by bake test results (Table In the case of the plain, chemical dough development (CDD) system, the time required for proofing is less affected for YTTS than for Strain A.

There was no effect on oven spring. In terms of 2) performance in the Sponge and Dough system, both strains behaved in a similar manner, although oven spring was less affected for YTTS.

The data of comparison 1 demonstrate that YTTS has a better tolerance to 37 0 C brew temperatures than Strain A.

This is manifested in terms of both yield and subsequent S'activity in a number of test conditions.

Comparison 2 A second important feature of YTTS is its keeping quality. The yeast samples examined in comparison 1 were 30 subjected to three standard keeping tests. These involved conducting 30 0 C plain dough activity tests before and after storage for 21 days at 4 0 C. The results of these analyses appear as Table 5. At both brew temperatures YTTS out-performed Strain A in terms of retention of activity on storage. This was particularly true of YTTS grown at 30 0 C where a very stable product can be produced.

8 Strain Characterisation Strain YTTS was characterised according to the method of van der Walt and Yarrow (pp 45-104, in The Yeasts: a taxonomic study, Kreger van Rij, Ed. Elsevier, Amsterdam, 1984) and Heard and Fleet (J.Appl. Bacteriol., 68: 447-451, 1990). The following characteristics were observed.

Growth in Malt Extract Broth (van der Walt and Yarrow): After 24 hours at 30 0 C the cells were globose '0 x (6.9-9.8)im. After 72 hours the cells measured x ;4.9-10.8)uLn.

Growth on MY Agar (van der Walt and Yarrow): After 72 hours at 30°C the colonies were circular, convex with an entire margin, cream in colour, dull and smooth.

Acetate Agar (van der Walt and Yarrow): ascospores were produced. 34% of cells had produced asci containing 1-4 globose spores after incubation for 72 hours at 25 0

C.

Fermentation Tests (Heard and Fleet): glucose, galactose, maltose, methyl alpha glucoside and sucrose 20 were fermented. Melibiose, lactose, cellobiose, inulin, starch and D-xylose were not fermented.

Assimilation Tests (Heard and Fleet): galactos;e sucrose, maltose, methyl alpha glucoside, raffinose, and melezitose were assimilated. L-arabinose, D-arabinose, D-rhamanose, cellobiose, melibiose, lactose, starch, erythritol, xylitol, D-mannitol, 2-keto-D-gluconate, 'D-gluconate, D-glucuronate, DL-lactate, succinate, citrate, ethanol, 2,3 butanediol, nitrate, nitrite ethylamine, lysine, cadaverine, creatine and creatinine 30 were not assimilated.

Other tests (Heard and Fleet): there was no growth in the presence of 100ppm cycloheximide or 1% acetic acid.

According to the description given by Yarrow (pp 379-395, in The Yeasts: a taxonomic study, Kreger van Rij, Ed. Elsevier, Amsterdam,1984) this yeast is identified as Saccharomyces cerevisiae Meyen ex Hansen (1883). This i identification is also supported by the data given by Barnett, Payne and Yarrow (Yeasts: characteristics and identification. 2nd ed., 1990).

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

o TABLE I Ferineiitograpli Formulations Used iii Activity Tests IngrC(Iieuts Plainl doughl 7% sugar 16%) sugar 25%Y sugar dough dough Clough Flour 280a 280g 250g 250g Sugar -19.6g 40g 62,5g Bread Improver* 1.5g 1.5g 1.3g 1.3g Water 1001111 lO0MI 91111l 92ml Salt Solution w/v) 57m]i 57rn1 26ml 26ml1 Yeast 30% solids) 5g 5g log tog Mauri Foods Bakerine Special o .0 C TABLE 2 Bake Test Formulations

COO

C.

*0 C C. CC

C

C

Plaiin Spongle anid Dough Ingredieut CDD Sponge Stage IDough Stage Flour 2000g 1400g 600g Sugar -160g Bread Improver 5*12g*- Water 1200inl 9001111 300in1 Salt 40g Yeast 30% solids) 60g Vegetable Oil 40g Emulsifier (SSL) Mauri Foods Bakerex Instant Mauri Foods Bakerine Special O 0

O

D D

O

D r Table 3 Yield and Activity Fermentograph Test (total ml CO, evolved per 2 hours) Strain Brew Temp Average 300C Test 370C Test Yield Plain 7% sugar 16% sugar 25% sugar Plain 7% sugar Strain A 300C 152 1240 1200 1145 450 2155 2000 37 0 C 130 1040 780 860 330 1750 1478 Loss 14.5 16 35 25 27 19 26 YTTS 30 0 C 168 1300 1165 1380 760 2020 1850 370C 151 1135 965 1260 600 1925 1700 Loss 10.1 13 17 9 21 5 8 All activities corrected to 30% solids and 52% protein Yield expressed as grams of wet yeast 30% solids) per 100g total reducing sugar I I 12 TABLE 4 Bake Test Comparison Baking'Test Strain Brew Yeast Plain CDD Sponge Dough Proof Time Oven Spring Proof Time Oven Spring (mrin) (mim) (ini) (Jili) Strain A 300C 65 30 58 370C 77 30 66 Loss 18 0 14 43 YTTS 300C 62 30 58 37 0 C 68 30 66 Loss 10 0 14 14 TABLE Keeping Quality Comparison Acivity Strain Brew Temp Initial After 21 days Activity Retention Strain A 300C 1240ml 915m1 74% ***37 0 C 1040m1l 845m1 8i% **YTTS 30 0 C 1300m1 1260rn1 97%* 37 0 C 1135m1 1025m1 *Volume Of CO 2 evolved in 2 hour plain Fermentograph test **Average of measurements at 24.5% solids and 28,5% solids (24.5% sofids-100%; 28,5%

Claims (3)

1. A baker's yeast Saccharomyces cerevisiae strain, the strain being characterised in that there is less than a 12% decrease in yield of grams of wet yeast at 30% solids per 100g total reducing sugar when the strain is brewed at 370C in comparison to brewing at 30 0 C and in that the strain retains at least 90% of its activity in plain dough at 370C when brewed at 37 0 C in comparison to when brewed at 300C and in that the strain brewed at 30°C retains at least 95%, preferably at least 97%, of its activity after storage for 21 days at 40C at 24.5% solids.

2. A baker's yeast Saccharomyces cerevisiae strain as claimed in aymma rf claimv 1 te in which the strain is YTTS (AGAL N93/10504).

3. A fresh or dry bakers yeast composition, the composition being characterised in that it includes bakers yeast Saccharomyces cerevisiae strain as claimed in any one claims 1 to 2. S SDATED this 4th day of July 1997 a. 25 BURNS PHILP COMPANY LIMITED Patent Attorneys for the Applicant: F.B. RICE CO 3 NT 9 *k 9 I I r ABSTRACT The present invention relates to new baker's yeast Saccharomyces cerevisiae strains which have improved temperature tolerance and/or keeping quality. An example of such a strain has been designated YTTS and deposited AGAL and accorded accession No. N93/10504. Goo* 096 S0.0