A method of processing spent brewer’s yeast

Field of the Invention

The present invention relates to a method of processing spent brewer’s yeast, and a spent brewer’s yeast product obtained by the method

Background to the Invention

Spent yeast biomass is the second most plentiful by-product of the brewing industry, with approximately 15-18 tons of surplus yeast produced per 10,000 hL of beer. Brewer’s spent yeast (BSY) serves well as an inexpensive source of protein, fibre vitamins, and minerals. With an increasing global interest in sustainability, the upcycling of waste materials for reuse is gaining momentum. However, due to its extremely bitter taste, there are very few outlets for BSY. The current primary use of brewer’s spent yeast is in formulations for animal and fish feed, where it has been shown to induce positive effects, including increased milk yield, improved pathogen resistance, and improved general health. Efforts have been made to debitter spent yeast but often require toxic alkali treatments, which is not ideal if the material is intended for food applications. Physical separation by means of rotary microfiltration has also been explored but was found to be not as effective as the alkaline washing methods.

Another challenge facing the increased use of spent yeast in food products is the high proportion of RNA present in yeast cells. RNA degrades into purines and uric acid, which can cause health issues such as hyperuricemia and gout in large volumes. Generally, there is no issue if yeast ingredients are incorporated at low levels. However, there could be health risks if yeast is used as a significant source of nutrition in the diet. Several methods of decreasing RNA content have been explored, comprising of chemical and autolytic methods, though few have been applied to spent yeast. The use of membrane separation technology to significantly reduce RNA content in spent yeast has been described. The reduction of RNA through processing technologies could allow for an increased volume of the processed BSY to be consumed more regularly, optimising the nutritional benefits of the BSY.

To date, applications for BSY are largely animal-feed based, and even in animal feed, the high bitterness, alcohol content, and susceptibility to spoilage can be challenging.

It is an object of the invention to overcome at least one of the above-referenced problems.

Summary of the Invention

The Applicant has addressed the problems of the prior by providing a method of processing that allows BSY to be transformed into a pleasant tasting, nutritious and functional ingredient which enables the ingredient to be used for human consumption. The process developed comprises autolysis of the BSY followed by lactic acid fermentation and optionally drying. The process reduces the bitterness drastically, reduces RNA and alcohol significantly, and changes the sensory profile as well as the techno-functional properties of the fermented BSY. The resultant fermented BSY product is very high in protein and valuable dietary fibre and therefore follows the current trend to increase the intake of plant and mycoproteins and fibre. Since the fermented BSY is produced from a waste stream of the brewing industry, it would be regarded as a sustainable process

In accordance with the first aspect of the invention there is provided a method of processing spent brewer’s yeast (BSY) comprising the steps of: autolysis of the BSY; and lactic fermentation of the autolysed BSY (typically with a lactic acid bacterium (LAB)) to provide a fermented BSY product.

In any embodiment, the step of lactic fermentation comprises incubating in a reactor vessel a fermentation mixture comprising autolysed BSY and a fermenting bacterium, typically a lactic acid bacterium.

In any embodiment, the fermentation mixture comprises autolysed BSY, a fermenting bacterium, and a sugar.

In any embodiment, the fermentation mixture consists essentially of autolysed BSY, a fermenting bacterium, and a sugar.

In any embodiment, the autolysed BSY makes up at least 60%, 70%, 80%, 90%, or 95% (w/v) of the fermentation mixture.

In any embodiment, the fermented BSY product is dried.

In any embodiment, drying is performed by freeze-drying or spray drying. Other drying methods, such as vacuum drying and rotary evaporation may also be applied.

In any embodiment, the fermented BSY product is pasteurised prior to drying.

In any embodiment, a carbon source is added to the BSY.

In any embodiment, the carbon source is added prior to autolysis.

In any embodiment, the carbon source is a simple sugar, such as sucrose, glucose or fructose. In any embodiment, the carbon source is added at 1-10%, 2-6%, 3-5% or about 4% (w/w).

In any embodiment, the LAB is an alcohol tolerant strain of LAB.

In any embodiment, the LAB is capable of growing in BSY having an alcohol content of up to 10% or 9%, and typically at least 3%, 4%, 5%, 6%, 7% or 8% (v/v).

In any embodiment, the LAB is a strain of Lactobacillus amylovorus.

In any embodiment, the strain of Lactobacillus amylovorus is Lactobacillus amylovorus FST 2.11 .

Lactobacillus amylovorus designated FST 2.11 is deposited under the accession no DSM 19280 on 13th Apr. 2007 at the DSMZ (Deutsche Sammlung von

Mikroorganismen and Zellkulturen). The invention is not restricted to this strain, and other L amylovorus strains, or indeed other lactobacillus strains showing antifungal activity and alcohol tolerance may be employed. Examples of other lactic acid bacteria that may be employed in the methods of the present invention include:

Lactobacillus plantarum FST 1.7

L paracasei FST 6.1

L brevis R2A

L rhamnosus LGG

Leuconostoc citreum TR116

L pseudomesenteroides MP070

Pediococcus pentosaceus YP1.7.1.1

In any embodiment, the autolysis step comprises thermal autolysis of the BSY. In any embodiment, the thermal autolysis comprises heating the BSY at for a time and temperature suitable to degrade yeast RNA, optionally followed by sterilisation to kill any viable yeast cells prior to the fermentation.

In any embodiment, the heating step comprises heating the BSY at 40-60°C for 12- 48h.

In any embodiment, the sterilisation step comprises sterilising the heated BSY at 80-100°C for 20-40 minutes.

In any embodiment, the sterilisation step comprises sterilising the heated BSY at 85-95°C for 25-35 minutes.

In another embodiment, the autolysis step comprises enzymatic autolysis.

In any embodiment, generally prior to the autolysis step, the BSY is treated to reduce the alcohol content, optionally by centrifugation. Other dewatering methods may be employed such as a filtration technology.

In any embodiment, the solids content of the alcohol depleted BSY is normalised by adding water.

In any embodiment, the solids content of the BSY is normalised to 10 to 60%, preferably about 20 to 60%, (w/v).

In any embodiment, the solids content of the BSY is normalised to 30 to 50% (w/v).

In any embodiment, the solids content of the BSY is normalised to 35 to 45% (w/v).

In another aspect, the invention provides an autolysed and lactic acid fermented BSY product. In any embodiment, the autolysed and lactic acid fermented BSY product is a food ingredient (e.g., for use in foods or beverages).

In another aspect, the fermented BSY product is obtained by a method of the invention.

In any embodiment, the fermented BSY product has a bitterness lower than BSY that has not been autolysed and subjected to lactic acid fermentation.

In any embodiment, the fermented BSY product comprises less than 129, 110 or 105 IBU mg/ L of bitterness compounds ((iso-a-acids) as determined using the method described below.

In any embodiment, the fermented BSY product has a RNA content lower than BSY that has not been autolysed and subjected to lactic acid fermentation.

In any embodiment, the fermented BSY product has a RNA content of less than 2%, 1 .5% or 1 .2% (w/w) as determined using the method described below.

In any embodiment, the fermented BSY product has a protein content that is at least 95% (w/w) of BSY that has not been autolysed and subjected to lactic acid fermentation.

In any embodiment, the fermented BSY product has a protein content of 25 to 40 % (w/w) as determined using the method described below.

In any embodiment, the fermented BSY product has an energy content of at least 250, 270, 280 or 290 kCal as determined using the method described below.

In any embodiment, the fermented BSY product has a free amino acid content of 5 to 15 g/100 g.as determined using the method described below. In another aspect, the invention provides a food or beverage comprising the fermented BSY product of the invention.

In another aspect, the invention relates to the use of the BSY product of the invention as an ingredient for foods or beverages, for example as a flavouring agent in a food or beverage product.

Other aspects and preferred embodiments of the invention are defined and described in the other claims set out below.

Brief Description of the Figures

Figure 1 : A schematic illustration of one embodiment of the method of the invention.

Figure 2: Free Amino Acid composition of CBSY and PBSY, expressed as g/100 g.

Figure 3: Comparison of aroma, taste, and flavour characteristics, plotted using the mean intensity rating for CBSY (solid line) and PBSY (dashed line).

Figure 4: Colour Comparison of BSY ingredients; CBSY (Left) and PBSY (Right).

Figure 5: The separation profiles of CBSY and PBSY emulsions, showing light transmission over the length of the sample with the top of the cell on the left and the bottom on the right.

Figure 6: Temperature sweep curve, showing the storage modulus (G’), the loss modulus (G”) and temperature. Curves are plotted using the average values of triplicate data. Figure 7: Tan 6 values for CBSY and PBSY, as a function of time, over the defined heating and cooling cycle.

Detailed Description of the Invention

All publications, patents, patent applications and other references mentioned herein are hereby incorporated by reference in their entireties for all purposes as if each individual publication, patent or patent application were specifically and individually indicated to be incorporated by reference and the content thereof recited in full.

Where used herein and unless specifically indicated otherwise, the following terms are intended to have the following meanings in addition to any broader (or narrower) meanings the terms might enjoy in the art:

Unless otherwise required by context, the use herein of the singular is to be read to include the plural and vice versa. The term "a" or "an" used in relation to an entity is to be read to refer to one or more of that entity. As such, the terms "a" (or "an"), "one or more," and "at least one" are used interchangeably herein.

As used herein, the term "comprise," or variations thereof such as "comprises" or "comprising," are to be read to indicate the inclusion of any recited integer (e.g. a feature, element, characteristic, property, method/process step or limitation) or group of integers (e.g. features, element, characteristics, properties, method/process steps or limitations) but not the exclusion of any other integer or group of integers. Thus, as used herein the term "comprising" is inclusive or open- ended and does not exclude additional, unrecited integers or method/process steps.

The invention will now be described with reference to specific Examples. These are merely exemplary and for illustrative purposes only: they are not intended to be limiting in any way to the scope of the monopoly claimed or to the invention described. These examples constitute the best mode currently contemplated for practicing the invention.

Materials and Methods

Raw material and bacterial strain

Spent yeast was provided by commercial brewery and frozen at -20°C until required. The yeast provided was a 5th generation lager strain. Yeast was cropped after ~120-160 hours of fermentation (5-7 days). Lactobacillus amylovorus FST2.11 was originally isolated from a cereal environment (Arendt et al., 2009) and is part of the University College Cork culture collection. A wide range of lactic acid bacteria strains were screened for their performance in a BSY fermentation and used to optimise the fermentation process (data not shown) before L amylovorus FST2.11 was selected for use due to its superior performance. Frozen stock cultures were maintained at -80°C in 40% glycerol. The strain was routinely cultivated on deMan- Rogosa-Sharpe (MRS) agar supplemented with 0.05g/L bromocresol green and incubated anaerobically at 30°C for 48h.

BSY fermentation

Prior to fermentation, the solids content of the BSY was standardised by centrifuging at 12,000 x g for 30 minutes, after which the supernatant was discarded, and sterile water was added to reach a solids content of 40%. 700ml aliquots of standardised BSY were placed into 1 L bioreactor vessels, to which 4% sucrose was added. Autolysis was performed at 50°C for 20h for degradation of yeast RNA, followed by sterilisation at 90°C for 30 minutes to kill any viable yeast cells prior to the fermentation. Working cultures of L amylovorus FST2.11 were prepared by inoculating single colonies into 10ml of MRS broth and incubating at 30°C for 24h, followed by subculturing (1 %) into fresh MRS broth for 18h. Cells were harvested by centrifugation at 5000 x g for 5 minutes, washed and resuspended in an equal volume of strength Ringer’s solution. After sterilisation, the BSY was cooled to 30°C and inoculated with 1 % washed L amylovorus FST2.11 cultures. Bioreactor fermentations were performed using a DASGIP Bioblock (Eppendorf, UK) for temperature and agitation control. BSY fermentations were carried out for 72 hours at 30°C with a stirring rate of 200 rpm. At the end of the fermentation period, the BSY was pasteurised at 72°C for 15 min (processed BSY; PBSY). BSY which did not undergo any standardisation or fermentation was pasteurised at 72°C for 15 min and included as a control (CBSY). PBSY and CBSY samples were frozen at -80°C and subsequently freeze-dried to produce a dried ingredient.

Characterisation of fermentation profiles

To monitor fermentation kinetics of FST 2.11 , samples were taken every 24h. The pH, TTA and microbial cell count were analysed.

2.3.1 pH, TTA, alcohol, and microbial growth determination

The pH and TTA of the samples were determined according to a standardised method (AACC 02-31.01 ). Alcohol content (% v/v) was measured using an Alcolyzer beer analysing system consisting of a DMA 4500 M density meter, an Alcolyzer beer ME measuring module and an Xsample 520 sample changer (Anton Paar, GmbH, Graz, Austria). RNA was isolated from the samples using a PureLink RNA Mini Kit (ThermoFisher Scientific, Massachusetts, USA) and quantified using a NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific, Massachusetts, USA)Cell counts of L. amylovorus FST2.11 were determined by diluting 1 ml aliquots of BSY in 9ml of strength Ringers’ solution. Samples were serially diluted and 10OpI aliquots were spread-plated onto MRS agar supplemented with 0.05g/L bromocresol green. Plates were incubated anaerobically at 30°C for 48h.

Dried ingredient characterisation

Compositional analysis

Compositional analysis was performed externally by Chelab SRL (Treviso, Italy): Moisture was determined gravimetrically; Protein was determined using the DUMAS method with a nitrogen-protein conversion factor of 6.25; fat was analysed using the Soxhlet method; Fibre was determined using Rapid Total Fibre Determination in accordance with AOAC 2017.16; Ash content was determined gravimetrically; Carbohydrate was determined by difference and energy values were determined by calculation; Total and free amino acid composition was determined using HPLC/ UV-Vis. Bitterness compounds (iso-alpha acids) were determined according to MEBAK method 2.17.1. RNA was isolated from the samples using a PureLink RNA Mini Kit (ThermoFisher Scientific, Massachusetts, USA) and quantified using a NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific, Massachusetts, USA)

Protein Profile

An Agilent Bioanalyser 2100 Lab-on-a-Chip capillary electrophoresis system ( California, USA) was used to determine the protein profiles of the materials and estimate the molecular weights of each of the protein bands. BSY samples were dispersed in 2% SDS, 2 M thiourea and 6 M urea, to give a protein concentration of 2 %. Dispersions were shaken for 2 h at 22 °C and centrifuged to remove insoluble material. Samples were analysed using an Agilent Protein 80 kit according to the instructions within the ranges of 5-80 kDa.

Aroma Compounds

Gas chromatography/ Olfactometry was completed externally by AromaLab (AromaLAB GmbH, Munich). To prepare sample for GC-O, 15 g of sample as extracted with 100 mL of diethyl ether. The organic layer was separated from the residue and the volatile compounds were isolated via distillation. The sample solution was dried over sodium sulphate and concentrated to 100 pL using a Vigreux column.

Sensory evaluation

The aroma, taste, and flavour characteristics of CBSY and FBSY were determined by a panel of seven experienced panel members. Sample preparation consisted of dissolving the CBSY and FBSY powders in water at a 5 % w/w concentration and labelling each with a three digit code to avoid bias. Characteristics included aroma, taste, flavour and aftertaste descriptors (Aroma; intensity, yeast, cereal/grain, beer, fruity, roasted. Taste; sour, bitter, sweet, umami, salty. Flavour; Intensity, mushroom, roasted, yeast, beer, cheese, fruity. Aftertaste; intensity). Samples were presented to the panel one at a time and the participants were asked to first rate the intensity of each attribute from 0 to 10 , with 10 being overwhelming and 0 being undetectable. Panellists were provided with water to cleanse the palate between samples, and a reference list defining each of the characteristics of interest was provided

Colour

The colour of the ingredient powders was measured using the Minolta colour measuring system (Chroma meter CR-400/410 Konica Minolta). Colour was measured using the CIE colour system (XYZ values) and then translated into and reported using the hunter colour system (L*a*b*).

Statistical analysis

All fermentations were performed in triplicate, while metabolomic analysis and sensory analysis were completed in duplicate. All were subjected to the independent samples t-test of statistical significance using the IBM SPSS software (IBM SPSS Statistics Ver. 28.0.0.0 (190) using a significance level of 5%. a- 3- and Total Glucans

The 1 ,3:1 ,6-[3-glucan and a-glucan content of the samples was determined using the Yeast and Mushroom [3-glucan assay kit (Megazyme, Bray, Ireland). This method consists of solubilising and hydrolysing all glucans ( 1 ,3:1 ,6-[3-D-Glucans,

1 .3-[3-D-glucans and a-glucans) in highly concentrated sulphuric acid. The remaining glucan fragments are then enzymatically hydrolysed to glucose, using

1 .3-|3- glucanase and [3-glucosidase. Glucose quantification using glucose oxidase/peroxidase (GOPOD) reagent, gives the value for total glucans.

Separately, a-glucans (i.e. phytoglycogen and starch) and sucrose are specifically hydrolysed using amyloglucosidase and invertase. [3-glucan is then determined by difference.

Foaming Dispersions (20 mL in 50 mL centrifuge tubes) with a concentration of 2% were prepared using distilled water. The pH was adjusted to pH 7 for half of the samples using a variety of HCI and NaOH dilutions. Samples were allowed to hydrate overnight at 4°C. After allowing the samples to return to room temperature, pH was checked and re-adjusted if needed. The samples were then frothed using an llltra- Turrax equipped with a S10N-10G dispersing element (Ika-Labortechnik, Janke and Kunkel GmbH, Staufen, Germany) at maximum speed for 30 s. The height of the foam phase was recorded immediately and after 60 mins. Foaming capacity and foam stability were determined by the following:

Foaming capacity (%) = (Foam height immediately after foaming I Initial sample height) x 100

Foam stability (%) = (Foam height after 1 h I Foam height immediately after foaming) x 100

Water and Oil Absorption

The water and oil binding capacities of the CBSY and PBSY were determined according to the method of (Boye et al., 2010) with some slight modifications. 15 mL tubes were weighed, and the weight noted and then 1 g of sample was weighed into each tube. Then 6 g of either sunflower oil or water was added to the tubes and mixed well, via vortex, for 3 minutes. Samples were then kept at room temperature for 1 hour. Samples were then centrifuged at 4000 x g for 30 minutes at 20°C. Supernatant was removed and samples were inverted and allowed to drain for 30 minutes. The tubes were then re-weighed and the % oil or water absorption was calculated using the following:

WHC/OHC (%)=((Weight of tube and pellet)-(Weight of tube)-(lnitial ingredient weight))/(lnitial ingredient weight) *100

Surface hydrophobicity

Surface hydrophobicity (SO) was measured based on the method of Hayakawa and

Nakai, using 1-anilino-8-naphthalenesulfonate (ANS), with slight modifications as described by Karaca et al. Protein dispersions of 0.2 % (w/v) protein were prepared in 0.01 M sodium phosphate buffer pH 7 and hydrated over night at 4°C, while shaking. Dilutions were prepared to give 8 ml in 15 mL tube (0.0006, 0.0012, 0.0025, 0.0050, 0.0100, 0.0150 % w/v protein). Following this, 2 mL of each dilution, and sodium phosphate buffer blank, was transferred to new 15 mL tubes and 20 pL of ANS (8.0 mM in 0.1 M phosphate buffer, pH 7) was added to 2 mL of each sample and they were left in darkness for 15 min. Fluorescence was measured (Aexcitation 390 nm, Aemission 470 nm) and adjusted with a blank measured without ANS. The results are presented as the slopes (r2 > 0.98) of the absorbance versus protein concentration.

Protein solubility

Protein solubility, at native and adjusted pH (pH 7) was evaluated using the Kjeldahl method. Firstly, the protein contents of CBSY and PBSY were determined. Following this, 1 % (w/v) protein dispersions were prepared and then left to hydrate over night at 4°C, while shaking. After returning to room temperature, pH was check ad re-adjusted if necessary. Samples were then centrifuged at maximum speed (4892 x g) for 30 mins. The protein content of the supernatant was determined, and protein solubility can be expressed as the % of total protein present in the supernatant.

Emulsion properties

The surface acting properties of CBSY and PBSY were examined by creating and analysing rough emulsions using the method of (Horstmann et al., 2017) with some modifications. A 1 % w/w aqueous protein solution was prepared using CBSY/PBSY and adjusted to pH 7 using NaOH and HCI. Samples were then hydrated overnight, shaking, at 4°C. These solutions were then used to prepare a 10 % w/w sunflower oil/protein solution mixtures where were then homogenised for 2 minutes using an Ultraturrax T10, fitted with S10N - 10G dispersing tool, to create an emulsion. Particle size was then analysed using a Mastersizer 3000 (Malvern Instruments Ltd., Worcestershire, UK), using a refractive index of 1.47. Emulsion stability was analysed using a LUMisizer (L.U.M. GmbH, Germany), at 100 ref for 15 minutes.

Rheological analysis Rheological tests were carried out using a rheometer (MCR301 , Anton Paar GmbH, Graz, Austria) equipped with a concentric cylinder measuring system (C-CC27- T200/SS, Anton Paar GmbH, Austria). Ingredient dispersions (20 % w/v) were hydrated overnight (16hrs) at 4 °C, while shaking. This concentration was determined by minimum gelling concentration pre-trials (data not shown). Small deformation oscillatory rheology was used to examine heat gelation, using a strain of 0.1 % and a frequency of 1 Hz. The temperature profile was defined as an increase from 20 °C to 90 °C, followed by a hold at 90 °C for 30 minutes, cooling to 20°C, and a hold at 20 °C for 30 minutes. This was followed by a frequency sweep from 0.01 Hz to 10 Hz, while maintaining 1 % strain.

Results

Table 1 Composition of control BSY and processed BSY, as analysed by CheLAB, internally (RNA) and TUM (a-acids):

CBSY PBSY g/100g D.M.

Moisture 6.7 ±0.4 14.5 ±0.38

Proteins 33.2 ± 1.4 (35.6 ± 1.501) 33.1 ± 1.4 (38.7 ± 1.638)

Total Fats 1.0 ±0.079 (1.4 ±0.001)

1.3 ±0.08 (1.4 ±0.089

Total dietary fibre 22.3 ±4.83 (23.9 ±5.177) 21.1 ±5.1 (24.7 ±5.968)

High molecular weight dietary 20.0 ±0.48 (21.4 ±0.515) 21.1 ±5.1 (24.7 ±5.968) fibre

Soluble dietary fibre 2.3 ±0.55 (2.4 ±0.59) <LOQ

Ash 5.3 ± 0.30 (5.6 ±0.322) 2.9 ±0.18 (3.4 ±0.211)

Total Carbohydrates 31.3 ±5.05 (33.5 ±5.416) 27 ±5.306 (31.8 ±6.209)

Energy value (kcal) 314 ± 10 294 ± 10

Energy value (kJ) 1322 ±45 1237 ±47 Dry matter 93.3 ± 0.38 85.5 ± 0.38

Calcium 0.2 ± 0.019 (0.249 ± 0.02) 0.270 ± 0.022 (0.316 ±

0.026)

Iron 0.007 ± 0.001 (0.007 ± 0.007 ± 0.001 (0.008

0.001) ±0.002)

Phosphorus 0.986 ± 0.079 (1.057 0.660 ± 0.053 (0.772 ±

±0.085) 0.062)

Magnesium 0.131 ±0.014 (0.153 ±

0.206 ± 0.023 (0.221 0016)

±0.025

Manganese 0.001 ± 0 (0.001 ± 0)

0.001 ± 0 (0.001 ±0) Potassium 1.490 ± 0.15 (1.597 0.495 ± 0.048 (0.579 ±

±0.161) 0.056)

Copper 0 ± 0 0 ± 0

Sodium 0.027 ± 0.003 (0.029 0.017 ± 0.002 (0.019 ±

±0.003) 0.002)

Zinc 0.007 ±0.001 (0.008 0.008 ± 0.002 (0.0.009

±0.002) ±0.002)

Total 2.956 (3.168) 1.588 (1.858)

Glucose 0.155 ± 0.013 (0.0166 ± 0 ± 0 (0.00 ± 0.00)

0.014)

Fructose 0.0836 ± 0.009 (0.09 8.48 ± 0.550 (9.924 ±

±0.009) 0.644)

Maltose 0.0421 ± 0.009 (0.045 0.106 ± 0.017 (0.124

±0.01) ±0.02)

Total Sugars 0.2807 ± 0.018 (0.301 8.586 ± 0.550 (10.048

±0.019) ±0.644)

RNA 4.256 ± 0.274 1.118 ± 0.269

IBU mg/ L

Bitterness compounds (iso-a- 129 100 acids)

Table 2: Total Amino Acid Profile of CBSY and PBSY, with results expressed as g/ 100g protein. Additionally, the percentage of essential amino acid requirement met per g protein is shown, as advised by the World Health Organisation (2007). * = Tyrosine is non-e

Essential CBSY % of PBSY % of

Amino Acids g/100 g requirement g/100 g requirement protein protein

Histidine 2.018 ± 0.331 134.5 1.964 ± 0.332 130.9

Isoleucine 3.524 ± 0.572 117.5 4.501 ± 0.695 150.0

Leucine 6.08424 ± 103.1 6.495 ± 0.816 110.1

0.81324

Lysine 7.560 ± 0.904 168.0 8.066 ± 0.967 179.2

Methionine 2.413 109.7 2.251 102.3 and cysteine Methionine 1.295± 0.208 80.9 1.489 ± 0.239 93.1

Cysteine and 1.117 ± 0.181 186.2 0.761 ± 0.124 126.9

Cystine Phenylalanin 6.747 177.5 7.25 190.8 e and tyrosine Phenylalanin 3.795± 0.633 4.0179 ± e 0.665

Tyrosine* 2.952 ± 0.482 3.232 ± 0.514

Threonine 4.428 ± 0.693 192.5 5.136 ± 0.725 223.3

Tryptophan 0.967 ± 0.105 161.1 0.810 ± 0.088 134.9

Valine 4.157 ± 0.663 106.6 5.649 ± 0.755 144.9

Non-essential

Amino Acids

Aspartic acid 9.217 ± 1.054 9.123 ± 1.057 Glutamic acid 14.729 ± 11.057 ±

1.566 1.208

Alanine 6.265 ± 0.813 6.132 ± 0.816

Arginine 6.054 ± 0.783 5.045 ± 0.725

Glycine 4.217 ± 0.663 4.018 ± 0.665

Proline 5.151 ± 0.723 4.803 ± 0.695

Serine 5.572 ± 0.753 5.498 ± 0.755

Total amino 87.017 ± 86.228 ± acids 3.142 3.051

Table 3: Gas Chromatography - Olfactory results; 0= not detected, 1= weakly detected, 2= unequivocally detectable, 3= intensely detectable

Compound Source Odour Flavour Intensity Intensity

-CBSY -PBSY

Ethyl-3- methyl Unknown Fruity, Sweet, 1.75 1.5 butanoate sweet, fruity, apple spicy

2,3,- Yeast/ Buttery/cr Buttery/cre 0.5 1.75 butanedione Bacterial eamy/sw amy/sweet

(diacetyl) fermentation eet

Acetic acid Bacterial Sour, Sour, 1.5 3 fermentation acidic acidic

2-ethyl-3,5/6 - Maillard Earthy/bu Earthy/bur 2 n.d. dimethyl reaction/pyroly mt nt pyrazine sis of serine and threonine Methional Thermal Boiled Boiled 2.75 2.25 induction/ potato, potato,

Maillard vegetabl vegetable reaction e

Butanoic acid Yeast cheesy, Sour, 2 1.5

(butyric acid) fermentation sharp, acidic, acetic cheesy

Phenylacetald Yeast green, honey, 1 2 ehyde fermentation sweet, floral, floral sweet, cocoa 2-/3- methyl Bacterial cheesy, cheesy, 3 2.5 butanoic acid metabolite sour, fruity, tropical fermented 2-acetyl-2- Yeast corn, com, 2 thiazoline fermentation potato, popcorn, toasted roasted, grain

Hexanoic acid Yeast Fatty, Cheesy, 2 metabolite - sour, fruity, formed during cheesy phenolic, butyric acid goat fermentation

Furaneol Maillard Caramel, Caramel, 3 reaction sweet, sweet, strawberr burnt y sugar, maple

Octanoic Acid Yeast fatty, soapy, 2 metabolite waxy, rancid, (under hypoxic cheesy, cheesy, conditions) fatty

4- Bacterial Phenolic, phenolic 1.5 methylphenol metabolite narcissus from amino , animal, acid mimosa breakdown

Lactic acid Bacterial Lightly Sour, n.d. metabolite Acidic acidic

Sotolon Yeast Caramel- Caramel- 2 metabolism like, like, sweet, sweet, maple maple Decanoic acid Yeast fatty, soapy, 2.5 2.25 metabolite rancid, waxy, (under hypoxic sour, fruity conditions) citrus phenylacetic Yeast honey, Honey, 2.5 2.25 acid metabolite sweet, sweet, floral floral vanillin Additive, beer vanilla, vanilla, 3 2.25 aging in wood, sweet, sweet, breakdown of creamy creamy phenol compounds during beer storage, wild contaminant yeast phenylpropioni Derived from floral, 2.25 2.5 c acid propionic acid, sweet, conjugate acid fatty of 3- phenylpropion ate

Value CBSY PBSY

L* 78.570 ± 0.240 69.709 ± 0.579 a* 2.479 ± 0.054 5.175 ± 0.111 b* 14.447 ± 0.232 18.347 ± 0.056

Table 5: Alpha-, Beta- and Total glucan values as determined for CBSY and PBSY, expressed as g/100 g dry matter g/100 g DM

Total glucan a-glucan p-glucan

CBSY 39.788 ± 0.443 14.822 ± 0.607 24.966 ± 0.753

PBSY 26.316 ± 0.693 11.319 ± 0.356 14.997 ± 0.379

Table 4 Foaming capacity (%) and foaming stability (%) of CBSY and PBSY after 30 and 60 minutes, at native pH and pH 7.

Native pH pH 7

CBSY PBSY CBSY PBSY

Foaming Capacity (%) 54.768 ± 36.296 32.308 ± 31 .282 ±

2.608 ±1.283 1.538 4.945

Foam stability (30 42.658 ± 0 ± 0.000 0 ± 0.000 69.006 ± mins) (%) 1.205 4.052

Foam stability (60 27.923 ± 0 ± 0.000 0 ± 0.000 57.627 ± mins) (%) 2.885 4.840

Table 5: Oil and Water absorption capacity of CBSY and PBSY, expressed as a percentage of initial sample weight

Fat Absorption Water Holding (%) (%)

CBSY 178.098 ± 1.379 52.704 ± 1.587

PBSY 183.310 ± 3.186 48.701 ± 0.321 Table 8: The surface hydrophobicity, protein solubility (%), and Zeta potential (mV) of CBSY and PBSY, at native pH and pH 7

CBSY PBSY

Surface Hydrophobicity 2906.933 ± 189.867 ±

(mV) 20.387 26.942

Protein Solubility (%) Native 31 .531 ± 0.420 75.426 ± 0.419 pH pH 7 31.508 ± 0.331 72.916 ± 0.174

Zeta potential Native -13.1167 ± 1 .532 -1 .17889 ± 0.301 pH pH 7 -14.1778 ± 1.525 -8.39333 ± 0.284

Summary

Fermentation with BSY significantly increases levels of free amino acids. The level of RNA in the food is decreased. Through processing, the flavour and aroma are transformed from bitter, yeast-like and alcohol-like to a more pleasant fruity, sour and floral profile. The levels of lactic acid and pyruvic acid are increased, and pH is lowered from pH 6 to pH3.5. The colour changed from a pale, creamy beige to a warm, biscuit-like brown. Protein solubility increased significantly. Foaming capacity and stability are improved. Many important functional properties, such as gelling, fat absorption and emulsifying properties are unaffected by the process. The key nutritional profile is unchanged by processing.

Equivalents

The foregoing description details presently preferred embodiments of the present invention. Numerous modifications and variations in practice thereof are expected to occur to those skilled in the art upon consideration of these descriptions. Those modifications and variations are intended to be encompassed within the claims appended hereto.