Field

The present invention relates to a method for manufacturing a yeast hydrolysate. Further the present invention relates to a yeast hydrolysate and the use thereof.

Background

The manufacture of yeast starts with a small sample of a pure culture. This sample is used to inoculate the first of a series of fermentors of successively increasing size. The first few are mildly aerated batch fermentations. In these stages, conditions are such that ethanol will be formed. Only the last two (or sometimes three) stages are performed using full aeration and incremental feeding of molasses. These fed-batch fermentations are carried out in fermentors of 100 m ³ (and more) net volume. Fermentation time is typically in the range of 12-20 hours, in which some 6,000 kg of fresh yeast is produced, on dry matter. After the feeding of substrates has stopped, aeration is usually continued at a reduced level for half an hour or so to let the yeast cells attain maturity and uniformity.

Further processing includes separation from the broth by centrifugation and washing which results in cream yeast (17-23 wt percent dry matter content). The cream yeast may be autolyzed for the production of yeast hydrolysates, such as yeast extracts and autolysates.

Yeast extracts and autolysates are produced by further treatment of cream yeast. Typically such a process comprises lysis of the yeast cells by a protease and optionally further treatment with phosphodiesterase for conversion of RNA into 5’ribonucleotides.

Yeast extracts and autolysates are known for their savoury and/or umami flavour profile, and its use is widespread in soups, sauces, ready to eat meals etc. There is a need in the art for yeast extracts and autolysates having advantageous flavour profiles, like having a liquorice type of flavour.

Further, there is a need for dark yeast extracts or autolysates, which advantageously provide a roasted flavour profile. Such dark yeast extracts or autolysates can be produced by allowing a Maillard reaction to happen, wherein the amino acids from the yeast extract or autolysate react with a reducing sugar.

There is a need in the art for improved production processes for dark yeast extract or autolysate. One aspect with Maillard reaction is the formation of acrylamide, as a result of heating the amino acid asparagine with a reducing sugar. There is a need in the art to provide production processes for dark yeast extract or autolysates with a reduced amount of acrylamide formation.

Detailed description It is an object of the present invention, amongst other objects, to provide a solution for above mentioned problems. Specifically, these problems, amongst other problems, are solved by the method according to the appended claim 1. More specifically, these problems, amongst other problems, are solved by a method for manufacturing a yeast hydrolysate, comprising the steps of: (i) providing a composition of unwashed yeast cells;

(ii) subjecting the unwashed yeast cells to autolysis;

(iii) concentrating the autolysed yeast cells to provide the yeast hydrolysate; wherein the autolysed yeast cells provided in step (ii) are maintained at a temperature above 60°C until concentrating step (iii). Surprisingly, the present inventors found that the method produces yeast hydrolysates that have a dark colour, and a liquorice type of flavour profile. Without wishing to be bound by any theory, it is expected that by using unwashed yeast cells, the yeast cells comprise solids remaining from the molasses used for cultivating the yeast. These solids are expected to contribute to the dark colour and liquorice type of flavour profile of the present yeast hydrolysate.

In a preferred embodiment, the present method further comprises the step of:

(iv) heating the yeast hydrolysate to a temperature above 80°C.

The advantage of heating the yeast hydrolysate is that a Maillard reaction occurs. This is beneficial in that a darker yeast hydrolysate is obtained, which also has more reacted or roasted flavour profile. Wthout wishing to be bound by theory, it is expected that solids remaining from the molasses used for cultivating the yeast contribute to the Maillard reaction.

Preferably, the yeast hydrolysates are heated to a temperature above 85°C, above 90°C, above 95°C, above 100°C, above 105°C, above 110°C, above 115°C, above 120°C, above 125°C or even above 130°C. Preferably, the yeast hydrolysate is heated to a temperature within the range of 85°C to 200°C, more preferably 90°C to 160°C, most preferably 95°C to 140°C. Preferably the yeast hydrolysate is heated to the indicated temperatures for a time period that allows a Maillard reaction to occur. Preferably, the yeast hydrolysate is heated to a temperature above 80°C for a time period of 1 to 20 hours, 2 to 15 hours, 3 to 12 hours, 4 to 10 hours or 5 to 9 hours. The present autolysed yeast cells provided in step (ii) are maintained at a temperature above 60°C until concentrating step (iii). The present inventors found that storage of the autolysed yeast cell walls at a temperature above 60°C until concentration provides a darker yeast extract, having a further reacted or roasted flavour profile. Without wishing to be bound by any theory, it is expected that maintaining the autolysed yeast cells at a temperature above 60°C until concentrating step (iii) suppresses growth of any microorganisms, such as Bacillus and lactic acid bacteria, and therefore avoids that glucose present in the autolysed yeast cells is utilized by these microorganisms. The glucose is advantageous to maintain because it contributes in the Maillard reaction. Preferably, the present autolysed yeast cells provided in step (ii) are maintained at a temperature above 65°C until concentrating step (iii), or above 70°C. Preferably, the present autolysed yeast cells provided in step (ii) are maintained at a temperature within the range of 60°C to 80°C, or 65°C to 75°C until concentrating step (iii).

In a preferred embodiment, the present composition of unwashed yeast cells is produced by separating yeast from a fermentation broth using centrifugation, without using washing steps. Preferably without using washing steps with water. Usually, after fermentation, yeast cells are separated from the broth and washed with water to provide the cream yeast. The present unwashed yeast cells differ from cream yeast in that the yeast cells are not washed. Unwashed yeast cells are different from cream yeast in that a supernatant obtained from the unwashed yeast cells has a dry matter within the range of 5% to 15%, whereas a supernatant obtained from cream yeast has a dry matter below 1%. In other words, the composition of unwashed yeast cells comprises solids that a cream yeast does not comprise due to the washing steps. Preferably, the present composition of unwashed yeast cells comprises solids other than solids belonging to the yeast cells.

Therefore, in a preferred embodiment, the present composition of unwashed yeast cells comprises solids remaining from molasses, preferably solids remaining from the molasses used for cultivating the yeast, more preferably solids chosen from the group consisting of betaine, raffinose, plant residues from sugar beet and plant residues from cane. Sugar beet and cane molasses are both conventionally used as substrate for cultivating yeast. In a preferred embodiment, the present composition of unwashed yeast cells is produced by cultivating yeast using sugar cane or beet molasses followed by separating yeast from the fermentation broth using centrifugation, without using washing steps. Betaine, or trimethylglycine, is a known residue from sugar beet. In a preferred embodiment, the present yeast hydrolysate provided in step (iii) has a colour of more than 40 AU.ml/g, preferably measured at 400 nm, on salt free dry matter. Preferably a colour of more than 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, or more than 300 AU.ml/g (measured at 400 nm) on salt free dry matter. Preferably, the present yeast hydrolysate provided in step (iii) has a colour of less than 1000 AU.ml/g, less than 900, less than 800, less than 700, less than 600 or less than 500 AU.ml/g, measured at 400 nm on salt free dry matter. Preferably, the present yeast hydrolysate provided in step (iii) has a colour within the range of 40 to 600, or 80 to 500 AU.ml/g, measured at 400 nm, on salt free dry matter. Preferably, the present colour is determined using the following test:

Absorbance is measured in clear sample solutions wherein 2 grams of yeast extract or autolysate is added to 100 ml demi water. 5 ml of the measuring solution is filtered twice over a filter of 0.2 pm (mesh size) to provide sample filtrates. A spectrophotometer blank (zero absorbance) is set using a 10 mm cuvette filled with water at 400 nm. The absorbance (A) is determined of the sample filtrates, subsequently the absorbance unit is calculated using the formula:

Absorbance unit [AU.ml/g] = A x (S + B) x D / (S x 1.005)

A = Measured absorbance at 400 nm B = Average water weight [g] D = Dilution factor

S = Sample weight [g]

1.005 = Density [g/ml]

Subsequently, the absorbance unit is expressed on salt free dry matter using the following formula: wherein absorbance unit is as defined above, wherein dry matter is the dry matter of the yeast hydrolysate, and the NaCI is the amount of NaCI in the yeast hydrolysate.

In a preferred embodiment, the present yeast hydrolysate has an amount of acrylamide of less than 200 ppb. Surprisingly, the present inventors found that acrylamide formation is low in the present method. This is advantageous in that acrylamide is a carcinogenic compound and is not desired in food or feed. Therefore, preferably, the present yeast hydrolysate has an amount of acrylamide of less than 180 ppb, preferably less than 160 ppb, preferably less than 140 ppb, preferably less than 120 ppb, preferably less than 100 ppb, preferably less than 80 ppb, preferably less than 60 ppb, preferably less than 40 ppb, preferably less than 20 ppb. Preferably, the yeast hydrolysate has an amount of acrylamide of more than 1 ppb, preferably more than 5 ppb, preferably more than 10 ppb, preferably more than 15 ppb.

In a preferred embodiment, the present yeast hydrolysate is a yeast extract or a yeast autolysate.

Food Chemical Codex defines a “yeast extract” as follows: "Yeast Extract comprises the water soluble components of the yeast cell, the composition of which is primarily amino-acids, peptides, carbohydrates and salts. Yeast Extract is produced through the hydrolysis of peptide bonds by the naturally occurring enzymes present in edible yeast or by the addition of food-grade enzymes".

The Food Chemical Codex defines Autolysed Yeast as follows: "Autoiysed Yeast is the concentrated, not extracted, partially soluble digest obtained from food-grade yeasts. Solubilization is accomplished by enzyme hydrolysis or autolysis of yeast cells. Autolysed Yeast contains both soluble and insoluble components derived from the whole yeast celf'.

In the context of the present invention autolysis of the unwashed yeast cells is defined as a process wherein degradation of the yeast cells is at least partially effected by active native microbial enzymes released in the medium after (partially) damaging and/or disrupting the microbial cell wall.

In a preferred embodiment, present step (iii) of concentrating the autolysed yeast cells to provide the yeast hydrolysate comprises a solid / liquid separation step, preferably a separation step to separate the soluble components of the yeast cell from the solids, to provide a yeast extract.

Alternatively, present step (iii) concentrating the autolysed yeast cells to provide the yeast hydrolysate does not comprise a solid / liquid separation step, to provide a yeast autolysate.

In a further preferred embodiment, the present step (iii) of concentrating the autolysed yeast cells to provide the yeast hydrolysate comprises an evaporation step. This evaporation step can be combined with a solid / liquid separation step, if a yeast extract is produced. Preferably, concentrating the autolysed yeast cells to provide the yeast hydrolysate provides a yeast hydrolysate having a dry matter content within the range of 30 to 60, preferably 40 to 50. The yeast cell can be any yeast cell. Preferably the yeasts is chosen from Saccharomyces, Kluyveromyces and Candida. Strains belonging to the genus Saccharomyces, in particular belonging to the species Saccharomyces cerevisiae are most preferred. Preferably, subjected the unwashed yeast cells to autolysis comprises damaging and/or partially disrupting the microbial cell wall enzymatically. A better control of the process can thereby be achieved and because this method is especially suitable to be used at large scale. Several enzyme preparations can be used like cellulases, glucanases, hemicellulases, chitinases, proteases and/or pectinases. Preferably protease is used, more preferably endoprotease is used. The conditions used to initiate the autolytic process are dependent on the type of enzyme used and can be easily determined by those skilled in the art. Generally the conditions used to enzymatically damage and/or disrupt the microbial cell wall will correspond to those applied during the autolysis of the microorganism. The autolysis of the yeast cells is at least partially effected by active native microbial enzymes released in solution after (partially) damaging and/or disrupting the microbial cell wall wherein the chemicals, or more preferably, the enzymes added to damage and/or to disrupt the microbial cell wall may contribute to the degradation of the microbial cells and of polymeric microbial material. Preferably, the conditions applied in the autolysis of Saccharomyces cerevisiae are such that the pH is between 4.5 to 9 and/or the temperature is between 45 to 65°C. Preferably the pH is 5 to 5.5 and at a temperature of 50 to 55°C.

Given the beneficial properties of the present yeast hydrolysate, the present invention relates, according to another aspect, to a yeast hydrolysate obtainable by the present method.

Preferably, the present invention relates to a yeast hydrolysate having a colour of more than 40 AU.ml/g, measured at 400 nm, on salt free dry matter.

Preferably, the present yeast hydrolysate has a colour of more than 40 AU.ml/g (measured at 400 nm) on salt free dry matter. Preferably a colour of more than 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, or more than 300 AU.ml/g (measured at 400 nm) on salt free dry matter. Preferably, the present yeast hydrolysate has a colour of less than 1000, less than 900, less than 800, less than 700, less than 600 or less than 500 AU.ml/g (measured at 400 nm) on salt free dry matter. Preferably, the present yeast hydrolysate has a colour within the range of 85 to 600, or 100 to 500 AU.ml/g, or 100 to 200 AU.ml/g (measured at 400 nm).

In a preferred embodiment, the present yeast hydrolysate has an amount of acrylamide of less than 200 ppb. Surprisingly, the present inventors found that acrylamide formation is low in the present method. This is advantageous in that acrylamide is a carcinogenic compound and is not desired in food or feed. Therefore, preferably, the present yeast hydrolysate has an amount of acrylamide of less than 180 ppb, preferably less than 160 ppb, preferably less than 140 ppb, preferably less than 120 ppb, preferably less than 100 ppb, preferably less than 80 ppb, preferably less than 60 ppb, preferably less than 40 ppb, preferably less than 20 ppb. Preferably, the yeast hydrolysate has an amount of acrylamide of more than 1 ppb, preferably more than 5 ppb, preferably more than 10 ppb, preferably more than 15 ppb.

According to another aspect, the present invention relates to the use of the present yeast hydrolysate in food or feed. Preferably wherein the food is a sauce, preferably oyster sauce.

The invention is illustrated in the examples below.

Examples

Materials and methods

Colour measurement

Absorbance is measured in clear sample solutions. 2 Gram of yeast extract is added to 100 ml demi water. 5 ml of the measuring solution is filtered twice over a filter of 0.2 pm (mesh size) to provide the sample filtrates. A spectrophotometer is calibrated with water in a 10 mm cuvette at 400 nm. The absorbance (A) is determined of the sample filtrates.

Absorbance unit is calculated using the following calculation:

Absorbance unit [AU.ml/g] = A x (S + B) x D / (S x 1.005)

A = Measured absorbance at 400 nm B = Average water weight [g] D = Dilution factor

S = Sample weight [g]

1.005 = Density [g/m!].

Subsequently, the colour is expressed on Salt Free Dry Matter (SFDM) as AU.ml/g, using the following formula: Absorbance unit[AU .ml / g] dry matter [%] NaCl[%]

100 )

Acrylamide measurement Sample pre-treatment 600 g dried and homogenized sample is extracted using 5 ml of milliQ water. 1 pg of internal standard ¹³ C3 acrylamide in solution (CIL) is added to the extract. After 10 minutes of centrifugation (6000 rpm), 3 ml of the upper layer is brought on an Extreluut- 3BT column (Merck). Using 15 ml of ethylacetate, acrylamide is eluted from the column. Ethylacetate is evaporated under a gentle stream of nitrogen down to approximately 0.5 ml.

Chromatographic conditions

The ethylacetate solution is analysed using gas chromatography. Separation is obtained using a CP-Wax 57 (Varian) column (length 25 m, internal diameter 0.32 mm, film 1.2 pm) and helium as the carrier gas with a constant flow of 5.4 ml/min. Split-less injection of 3 pi is performed. Oven temperature is kept at 50°C for 1 minute, after which the temperature is increased with 30°C/min towards 220° C. After 12 minutes of constant temperature of 220°C the oven is cooled down and stabilized before next injection.

Detection is performed using on-line chemical ionization mass spectrometry in positive ion mode, using methane as ionization gas. The characteristic ions m/z 72 (acrylamide) and m/z 75 ( ¹³ C3 acrylamide) are monitored for quantification.

Example 1

Preparation of a dark yeast extract Saccharomyces cerevisiae yeast was cultivated by use of molasses from sugar beet. Thereafter, the yeast was concentrated by centrifugation to a concentrated cream yeast having a dry matter around 25%. No washing steps on the yeast cream (or on the fermentation broth) are carried out. Subsequently, two litres of cream yeast were heated to 53°C. Subsequently 2.5 ml Alcalase (commercially available serine protease from Novozymes, Denmark) was added and the mixture was incubated for 4 hours at pH 5.3 and 53°C. The conditions were adjusted to pH 5.1 and 51.5 °C and incubated for 12 hours to provide the autolysate. Thereafter, the autolysate was centrifuged to discard the cell walls and evaporated to a dry matter of 40 to 45%. Example 2

Preparation of a dark yeast extract

The yeast extract of example 1 was heated to 90°C for a time period of 8 hours to allow a Maillard reaction to occur.

Example 3

Preparation of a dark yeast extract

The method of example 1 was repeated, however after autolysis the autolysate was immediately stored at 65°C until centrifugation and thereafter evaporation. Thereafter, the concentrated yeast extract was heated to 90°C for a time period of 8 hours to allow a Maillard reaction to occur. The yeast extract was further tasted in a 1% wt. solution of the yeast extract in water, and provided liquorice and dark meat flavour notes.

Example 4

Colour measurement

The yeast extracts of example 1 , 2 and 3 where analyzed on dark colour, using the assay described above. The results are shown in table 1 :

Table 1.

The results in table 1 show that using unwashed yeast cells a dark yeast extract has been provided. Further, example 2 shows that heating the yeast extract to allow the Maillard reaction to happen results in a darker yeast extract. Further, example 3 shows that it is advantageous to maintain the autolysate at a higher temperature to avoid utilization of any glucose present, and results in a darker yeast extract.

Example 5

Acrylamide measurement

The yeast extracts of example 1 , 2 and 3 where analyzed for the presence of acrylamide, using the assay described above. The results are shown in table 2:

Table 2.

Example 6

Sensory study in oyster sauce

The yeast extract of example 3 was tested in oyster sauce. Commercial soy sauce was used (Liu Yue Xian Premium Original Soy, Shinho), and the yeast extract of example 3 was added to the soy sauce in an amount of 0.1 % (wt). The control is the oyster sauce as such. The products were tested using a sensory panel. The panelists rated the flavour attributes salty, umami, sweet, molasses/caramel, roast and complexity. The results are shown in figure 1. Figure 1 shows that the yeast extracts provides as advantageous flavour profile, with characteristics in roast, complexity and salty notes.