Role Of Casein Hydrolysate Manufacturing Conditions Biology Essay

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Milk Proteins Milk contains approximately 30-35g protein /L. Approximately 80 of which are present in casein micelles, these are large spherical complexes containing 92% protein and 8% low molecular mass components, mainly inorganic salts, principally calcium phosphate. Caseins represent four gene products αs1-casein, αs2-casein, β-casein and κ-casein. Figure 1.1.1 shows the different protein types and their concentrations in milk. (Advanced food Chemistry A)

The precise structure of the casein is micelle has been subjected to numerous scientific studied. Several models have been proposed over the last number of years in order to describe casein micelle behaviour. Caseins have distinct areas of positively and negatively charged groups in their primary structures resulting in amphiphilic properties. The caseins are known as rheomorphic proteins as they have extremely flexible molecular structure. The caseins are thought to have α-helical or β- sheet structures, again this is only from theoretical studies - no such structures have been found in the caseins because they have yet to be successfully crystallised. The secondary structure is loose and lacks order due to the high number of proline residues which cause the protein chain to bend in a particular way. Casein micelles are very stable against heat denaturation. As there is no tertiary there is considerable exposure of hydrophobic residues, these result in strong association reactions and makes them relatively insoluble in water. All the caseins are conjugated proteins, most with one or more phosphate groups which are esterified to serine residues. These phosphate groups are important to the structure of the casein micelle as calcium binding of the caseins is proportional to the phosphate content. Table 1.1.2 shows some of the physicochemical characteristics of the casein micelles. (University G)

Physicochemical Characteristics of Casein Micelle

Diameter

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50-300nm

Surface Area

8 x 10-10 cm2

Volume

2 x 10-15 cm3

Density

1.063 g cm-3

Molecular weight (hydrated)

1.3 x 109 Da

Voluminosity

4.4cm3 per g protein

Hydration

2g H2O per g protein

Water Content (hydrated)

63%

(Handbook) (Table 1.1.2)

Sodium Caseinate Manufacture

Separation of cream

Milk

Skim Milk

Rennet or Acid coagulation

(Heating and Washing)

Whey

Casein

Neutralisation

Caseinate

Enzymatic Hydrolysis

Peptides

(Fig 1.2.1) (Handbook)

Figure 1.2.1 gives a basic representation of the manufacturing process of sodium caseinate. The process involves firstly the separation of milk and cream, followed by pasteurization. The caseins are then precipitated either by the addition of a coagulant such as rennet or by a reduction in pH to 4.6 (its isoelectric point). The coagulated protein is heated to form a curd. The curd is then separated from the whey by filtration or centrifugation in combination with counter-current washing with water. The curd is then reacted with an alkali, eg sodium hydroxide and then dried to form a caseinate. (Handbook)

Bioactive Peptides

The primary structure of proteins consists of certain amino acid sequences that have the ability to exert physiological benefits in human beings. The amino acid sequences remain inactive when they are present as part of the continuous primary structure of the parent protein. However, when the parent protein is acted upon by an appropriate enzyme, the peptide is released (Dr Rotimi Aluko). Enzymatic hydrolysis of milk proteins has been shown to reduce antigenicity, and increase biological activity for example by the release of immunomodulating, opioid and antihypertensive peptides. To this end, growing interest has been focused on physiologically active peptides derived from milk proteins. In addition, the small peptides present in protein hydrolysates are absorbed more rapidly from the intestine than free amino acids or intact proteins. (Spellman et al). A summary of bioactive peptides which are derived from milk proteins and their functions are displayed in figure 1.3.1

(Hannu K) (Fig 1.3.1)

Enzyme preparation

The enzyme preparation used for casein hydrolysis in this study was Prolyve 1000â„¢ a commercially available proteinase preparation which is of bacterial origin. This preparation contains the enzyme Subtilisin Carlsberg which is from a family of serine endopeptidases isolated from Bacillus licheniformis. Endopeptidases are enzymes which cleave within the protein or polypeptide chain. Subtilisin Carlsberg has a broad specificity for the hydrolysis of peptide bonds, with a preference for a large uncharged residue. It is an aromatic enzyme with a preference for carboxyl side cleavage. Hydrolyzes peptide amides containing leucine and tyrosine residues. This enzyme preparation has been used in the hydrolysis of whey proteins (Spellman et al, 200 ) however to date there does not appear to be any publications using this enzyme activity in the hydrolysis of casein substrates.

Bitterness

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The main disadvantage of protein hydrolysis is bitterness. Intact food proteins due to their molecular size are unlikely to interact with the taste-bud receptors and as such would not contribute significantly to flavour (Enzymology). As hydrolysis breaks down these proteins into much smaller peptides interaction with taste-bud receptors can occur. Also in Intact casein micelles the most hydrophobic amino acids are oriented towards the interior of the molecule, during hydrolysis peptides containing hydrophobic amino acids are released. As hydrolysis continues, more hydrophobic amino acid residues become exposed for this reason hydrolysate bitterness generally increases with increasing hydrolysis (Spellman et al). The 'Q-rule' devised by (Ney, 1971)established a quantitative relationship between the amino acid composition of a peptide and its bitterness. Using the values calculated by (Tanford,1962), the Q-rule stated that peptides with an average hydrophobicity (Q) value greater than 1400 cal mol−1 and with molecular masses below 6000 Da elicit a bitter taste.(Lemieux,1992). Figure 1.5.1 shows several protein types and their Q values, it also shows the different casein classifications and their individual Q values. β-casein has a Q value of greater than 1400 kcal mol-1 this may somewhat predispose to bitterness if during the hydrolysis the proteins are broken down to peptides weighing less than 6000Da

(Figure 1.5.1)

Bitter peptides typically contain 3-15 amino acids and are characterised by the presence of hydrophobic amino acids such as leucine, isoleucine, proline, valine, phenylalanine, tyrosine and tryptophan. (Enzymology). The distinct bitter flavour of protein hydrolysates has been a major limitation in their use in food and health products, they would need to be incorporated into foods at very low concentrations to prevent its presence producing an unacceptable flavour.

Enzymatic Hydrolysis of protein and the factors which affect it

Enzymatic hydrolysis of protein is the process by which proteins are broken down by proteases. Several factors affect the rate hydrolysis these include, enzyme specificity, extent of protein denaturation, enzyme: substrate ratio, total solids concentrations, viscosity, pH, ionic strength, temperature and absence or presence of inhibitory substances.

The specificity of an enzyme is a key factor, influencing both the number and location of the peptide linkages that are hydrolyzed. Endopeptidases cleave the peptide linkage between two adjacent amino acid residues in the primary sequence of a protein, yielding two peptides. Proteolysis can proceed either sequentially, by releasing one peptide at a time, or through the formation of intermediates that are further hydrolyzed to smaller peptides. (Panyam et al, 1996).

There is very little information available about the effect of total solids on the rate of hydrolysis or the resulting properties of the hydrolysate samples. Spellman et al, 2004 carried out a study on whey protein hydrolysates and how total solids affected the rate of the hydrolysis and the physiochemical properties of the resultant hydrolysates. They concluded that the bitterness of the hydrolysate samples decreased with increasing total solids concentrations.

Viscosity is the measure of resistance to flow. 'Solvent viscosity can influence rates of enzyme catalyzed reactions by two principle mechanisms: (1) Since molecular diffusion coefficients vary inversely with the viscosity of the medium, an increase in solvent viscosity will lead to a decrease in the association rate of an enzyme and substrate. This will manifest itself in a viscosity-dependent decrease in kc/Km for reactions in which the process that is governed by kc/Km is diffusion-controlled. (2) Since solvent viscosity dampens structural fluctuations of proteins through frictional effects, increases in solvent viscosity will lead to decreases in reaction rates for catalytic processes that are dependent on enzyme structural fluctuations' Dang, 1998 this is supported by studies from Gavish, 1979 and Ng, 1991

The effect of temperature on the rate of enzymatic hydrolysis relates to the enzyme and the optimum conditions to facilitate its reaction but also to the substrate where caution must be taken in order to avoid /facilitate heat denaturation. Whichever of the conditions is preferred should be controlled.

pH also affects the rate of enzyme action and may pose a threat of denaturation the enzyme. pH also has an effect on protein solubility. The iso-electric point is the pH at which the protein has no net charge, at this pH proteins would lose solubility and precipitate out of solution. Enzymes work better on proteins in solution.

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The ionic strength of the may affect the rate of hydrolysis. A solution of low ionic strength (0.5M - 1.0M ions of neutral salts) may increase the solubility of the protein by 'salting in'. Whereas concentrations above 1.0M may reduce the solubility of the protein as it causes increased competition for water molecules, favouring protein- protein interactions and thereby 'salting out'.

TNBS Assay

The method used to quantify the degree of hydrolysis (DH) of the sodium caseinate hydrolysates was the trinitrobenzenesulfonic acid (TNBS) assay that was described by Adler-Nissen ,1979). Degree of hydrolysis (DH) is defined as the percentage of the total number of peptide bonds in the protein which have been cleaved by hydrolysis. The TNBS Assay was used in this project as it has been proven to be a highly accurate method for quantifying the DH of hydrolysate samples (Spellman, 2003). The one drawback of this method is that is requires long incubation and cooling steps.

This method is a spectrophotometric assay of the chromophore formed by the reaction of the TNBS with primary animes.

Figure 1.8.1 is a representation of the basic mechanism of how the TNBS assay works. Simply put the TNBS reagent binds to the NH2 group of the peptides in the sample and in doing so creates a yellow colour. The more hydrolyzed the sample is the more peptides there are and therefore the more NH2 groups to bind to and the more yellow the solution will become. These solutions are then read spectrophotometrically

.

This assay consists of several steps firstly the protein hydrolsate is dispersed in hot 1% sodium dodecyl sulfate (SDS), this serves to ensure an accurate result as it prevents clumping of the protein and enables the TNBS reagent to cleave all of the NH2 groups. This reaction favours slightly alkaline conditions (pH 8.2) which is facilitated by the addition of sodium phosphate buffer. TNBS reagent reacts slowly with hydroxyl ions and could cause the blank to give a false slightly increased reading. This increase is stimulated by light and for this reason many of the steps are carried out in the dark, such as the incubation for 1hour at 50°C and the subsequent termination of the reaction which is achieved by lowering the pH. Termination is accomplished by the addition of HCL, caution must be taken to ensure that the pH does not drop below 3.5 as this would causes turbidity. The samples are allowed to cool at room temperature for 30mins, cooling below room temperature may also cause turbidity. After standing for 30mins the samples and their absorbance readings are stable and more accurate.

Reverse Phase -High Performance Liquid Chromatography (RP-HPLC)

Reverse-phase (RP )- HPLC is an essential tool in the separation of proteins and peptides. RP-HPLC is widely used in protein studies because of its versatility, sensitive detection (can separate proteins of nearly identical structure) and its ability to work together with techniques such as mass spectrometry.

High performance liquid chromatography severs to enhance detectability of the analyte and can be applied to the analysis of any compound with solubility in a liquid that can be used as the mobile phase (Rounds, 1988). The major components of a high performance liquid chromatography system include a pump, injector, column, detector and data system. Reverse phase -HPLC is where the polarities of the stationary phase and the mobile phase are reversed in comparison to the normal phase of absorption chromatography (Macrae, 1988).

The stationary phase is a solid support that is non-polar. Reversed phase media are composed of a base matrix to which organic ligands for coating silica such as octyl (C8) or octadecyl groups (C18) are attached( Sofer, 1997). The mobile phase is a polar liquid that flows over the stationary phase. The sample is dissolved in the initial mobile phase (eg. Trifluoroacetic acid) prior to being filtered and applied by injection to the column. Polar mobile phases are usually water mixed with methanol, acetronitrile or trifluoroacetic acid (Rounds, 1988). The interaction of the components bring separated and the stationary phase rely on hydrophobic interactions and this determines the degree of migration in the column and separation of the components in the sample. Polar compounds are the first to be eluted as they are hydrophilic and have week interactions with the stationary phase.

The pumps function is to deliver the mobile phase through the system at a controlled flow rate of 1ml/min. Gradient elution system is used which involves two independent programmable pumps that are mixed at high pressures (Rounds, 1988). This allows different compounds to be eluted by increasing the strength of the organic solvent in a linear fashion. The use of a valve injector places the sample for separating into the following mobile phase and it is carried in this liquid for introduction into the column. The sample injection is usually automated. The HPLC column is usually constructed of stainless steel tubing with terminators that allow it to be connected between the injector and detector of the system (Rounds, 1988). The packing material for this column is in the form of a chromatographic bed and acts as both a stationary phase and a support.

The detector for the HPLC is the component that emits a response due to the eluting sample compound/ concentration changes in the column eluent and subsequently signals a peak on the chromatogram. Peptides do not absorb light above 220nm and absorption of 214nm may be used to follow the concentration of peptides in the column effluent (Sofer, 1997). Absorbance of peptide at this wavelength is performed as peptides do not have a three-dimensional structure and all the amino acids are exposed and easily interact with the chromatographic media. Proteins don not have the same interaction with the media as only a small quantity of its molecules tend to interact. Proteins normally show an absorbance at 280nm due to the content of aromatic amino acid substitutes (Sofer, 1997). Ultra violet detection allows tracing of protein concentration of the effluent and a chromatogram visually shows the peaks of the peptides and aromatic amino acids present in sample.

Column and mobile phase temperature and pH can affect the separation of proteins and peptides by RP-HPLC. Increasing the temperature reduces the retention of peptides. The temperature affects the relative retention of selectivity, which affects resolution (David Carr).

Sensory Evaluation

Sensory evaluation involves both principles of experimental design and statistical analysis. Sensory evaluation of food can use the human senses of taste, touch, sight and smell to evaluate different aspects of food such as flavour, texture, appearance and aroma. For this project the emphasis is on flavour and taste will be the sense of choice. Flavour can be referred to as the sensation perceived from food or liquid taken in the mouth (Fisher et al, 1997). The four basic tastes are sweet, sour, salty and bitter. For this project a sensory panel was selected and trained. Candidates for the panel were selected firstly on the basis of being able to distinguish between sweet, sour, salty and bitter. Successful candidates were then trained to detect and quantify bitterness using caffeine standards. Statistical analysis of the bitterness scores was carried out using the statistical programme R version 2.10.1©, One-way analysis of variance (ANOVA) and independent-samples t-tests were performed on sensory data. A significant result was defined as P < 0.05, a highly significant result was defined as P < 0.001. Sensory evaluation is a crucial aspect in every project whose ultimate goal is to market a food/ functional food product. Bitterness is a major limiting factor in the production and incorporation protein hydrolysate into foods and sensory evaluation is the most effective method of determining the bitterness level.

Objectives of this study

The objectives of this study are:

To generate sodium caseinate hydrolysates at different protein/ total solids concentrations.

Physicochemical characterisation of the hydrolysate samples.

Selection and training of a sensory panel to determine the level of bitterness of the hydrolysates.

To determine if the bitterness of sodium caseinate hydrolysates is related to protein/ total solids concentration at which the hydrolysates were generated.

Chapter 2

Materials and Methods

Materials and Methods

2.1 Materials

Sodium caseinate was obtained from Kerry Group, Ireland and its protein content was determined to be 88.01% using Kjeldhal analysis. Prolyve 1000 was obtained from Lyven Enzymes Industrielles ,Caen, France Trifluoroacetic acid (TFA), HPLC grade acetonitrile, L-leucine, HPLC grade water, Citric acid, sodium chloride, sucrose and caffeine were obtained from Sigma Chemical Co. (Poole, Dorset, UK). PuradiscTM 25 AS disposable syringe filters (0.2 µm), Supor_ hydrophilic membrane filters (47 mm, 0.2 lm) and 2N NaOH (Titripur, SWR, 1.09136,1000) were obtained from VWR chemicals, Ireland.

2.2 Protein determination using the Kjeldahl procedure

The Kjeldhal method for determining protein concentration is an accumulation of several reactions, the first of which is digestion, where protein nitrogen is liberated to form ammonium ions. Sulphuric acid oxides organic matter and it combines with the ammonium formed. The second reaction consists of the sulphuric acid in the sample being neutralised with NaOH forming ammonia which is then distilled into a 4% boric acid solution for the third reaction. For the forth reaction a titration takes place between the borate ions formed and the standardised 0.1M HCL until a pH of 4.6 is reached.

Øž Moles of HCL= Moles of NH3 = Moles of nitrogen in sample.

% Nitrogen x conversion factor (6.38) = % protein

Approximately 0.2g of sodium caseinate powder was accurately weighed out and transferred into a Kjeldhal flask. Sucrose was used as a blank. Into each flask 20mL of concentrated H2SO4 (Low in nitrogen) and two kjeldhal tablets. The samples were then placed in the Kjeldhal digestion unit (Buchi, Labortechnik AO, Postfrach, Switzerland) at 120°C for half an hour and temp increased to 420°C for 2 hours. During this time digestion of organic matter in the sample occurs. After digestion the tubes are then cooled before being transferred to the Kjeldhal Buchi B323 Distillation unit for distillation and subsequent determination of protein content. This procedure was performed in triplicate.

2.3 Enzymatic hydrolysis of sodium caseinate

For the remainder of this thesis the hydrolysate samples will be referred to in regards to protein concentration. Table 2.3.1 shows protein concentration in proportion to total solids concentration for the sodium caseinate hydrolysates generated at different protein/ total solids concentrations.

Table 2.3.1

Protein Concentration

Total Solids Concentration

5% (w/v)

56.8g/L

10% (w/v)

113.6g/L

15% (w/v)

170.4g/L

20% (w/v)

227.2g/L

The enzymatic hydrolysis experiments were carried out in a 2L sealed reaction vessel (___). The aqueous solutions of sodium caseinate were allowed to hydrate for ~ 2hours at 50°C with the aid of an overhead stirrer. The solutions were then stored in a fridge at 4°C overnight. On the day of the hydrolysis the solutions were equilibrated to 50°C and the pH was then adjusted to 7 by the addition of 2N NaOH before the addition of the enzyme. Prolyve 1000 was added at an enzyme: substrate (E:S) ratio of 25ml enzyme preparation / kg protein. The E:S ratio was estimated on the basis of what has previously worked for whey, the standard addition was 0.25mls of enzyme solution per 100ml to 10% solution. The solution was mixed with an over head stirrer (Heidolph

Instruments, Schwabach, Germany) and the pH was kept constant throughout the hydrolysis using a pH stat (718 Stat Titrino, Metrohm, Herisau, Switzerland). Hydrolysate samples were taken at various time intervals, quickly brought to 80°C using a microwave and then maintained at 80°C for 20mins in a water bath to inactivate the enzyme. The samples were then stored at -20°C until required for analysis.

2.4 Quantification of Degree of Hydrolysis (DH)

The TNBS reagent was made up of 0.1% (w/v) TNBS in water. 1% (w/v) SDS was used as the blank and l-Leucine was used as the standard. Samples and standard solutions were prepared in 1% (w/v) SDS. The hydrolysate samples were diluted (1 in 51, 1 in 76, 1 in 101, 1 in 126, 1 in 151 & 1 in 201 in accordance with their protein concentrations). All samples were done in triplicate. 0.25 mL of the test, intact sodium caseinate (control) and l-Leucine standard solutions was added to test tubes containing 2.0mL of sodium phosphate buffer (0.2125 m, pH 8.2). The following steps are light sensitive and took place in the dark: 2 mL of TNBS reagent was then added to each tube, followed by vortexing and incubation at 50°C for 60 min in a covered water bath. After incubation, the reaction was stopped by the addition of 0.1N HCl (4.0 mL) to each tube. The samples were then allowed to cool in the dark at room temperature in order to stabilize the absorbance readings. The absorbance values were then read at 340nm using a (Carey 100 double beam spectrophotometer)

The DH was calculated as follows:

Where the nitrogen content of peptide bonds = 112.1 mg of Nitrogen /g of protein for casein substrates. The protein concentration became marginally more dilute as NaOH was added throughout the hydrolysis reaction; the protein concentration was calculated accordingly.

2.5 RP-H.P.L.C.

Reversed-phase - HPLC was carried out on the sodium caseinate hydrolysate samples using a Waters HPLC system, comprising a Model 1525 binary pump, a Model 717 Plus autosampler and a Model 2487 dual λ absorbance detector interfaced with a BreezeTM data-handling package (Waters, Milford, MA, USA). The column used was a Phenomenex Jupiter (C18, 250r4.6 mm ID, 5 mm particle size, 300A° pore size) separating column (Phenomenex, Cheshire, UK) with a Security GuardTM system containing a C18 (ODS) wide pore cartridge (4r3 mm ID, Phenomenex, Cheshire, UK). The column was equilibrated with solvent A (0.1% TFA) at a flow rate of 1.0 ml min-1 and peptides were eluted with an increasing gradient of solvent B (0. 1% TFA, 80% acetonitrile). TFA is used in HPLC as it improves symmetry of signals. Detector response was monitored at 214 nm & 280nm. The sodium caseinate hydrolysate samples were diluted to 0.8% (w/v) in 0.1% TFA, filtered through 0.2 µm syringe filters and 20 µl was applied to the column. The following tables show the 3 different gradient profiles used in analysing the samples.

Basic reverse phase high performance liquid chromatography gradient profile

Time

Flow

%A

%B

Curve

1

0.01

1.00

100.0

0.0

6

2

4.00

1.00

100.0

0.0

6

3

54.00

1.00

40.0

60.0

6

4

55.00

1.00

0.0

100.0

6

5

65.00

1.00

0.0

100.0

6

6

70.00

1.00

100.0

0.0

6

7

85.00

1.00

100.0

0.0

6

(Table 2.5.1)

Modified Profile 1

Time

Flow

%A

%B

Curve

1

0.01

1.00

100.0

0.0

6

2

4.00

1.00

100.0

0.0

6

3

84.00

1.00

40.0

60.0

6

4

85.00

1.00

0.0

100.0

6

5

95.00

1.00

0.0

100.0

6

6

100.00

1.00

100.0

0.0

6

7

115.00

1.00

100.0

0.0

6

(Table 2.5.2)

Modified Profile 2

Time

Flow

%A

%B

Curve

1

0.01

1.00

100.0

0.0

6

2

4.00

1.00

100.0

0.0

6

3

114.00

1.00

40.0

60.0

6

4

115.00

1.00

0.0

100.0

6

5

125.00

1.00

0.0

100.0

6

6

130.00

1.00

100.0

0.0

6

7

145.00

1.00

100.0

0.0

6

(Table 2.5.3)

The ultimate profile used on the samples with similar DH values but of different protein concentrations was the modified profile 2.

2.6 Sensory

In selecting candidates for the taste panel a recognition test for the four tastes was firstly performed. This involved making up solutions of citric acid monohydrate (sour), sucrose (sweet), caffeine (bitter) and NaCl (salt) all of which were made up in Ballygowan still water. At least two concentrations of each sample representing a taste were included in the sensory evaluation see table 2.6.1

Recognition Test Layout

Sample Letter

Concentration

Chemical (Taste)

A

0.02% (w/v)

Citric acid monohydrate (Sour)

B

0.40%(w/v)

Sucrose (sweet)

C

0.03%(w/v)

Citric acid monohydrate (Sour)

D

0.02%(w/v)

Caffeine (Bitter)

E

0.08%(w/v)

NaCl(Salty)

F

0.60%(w/v)

Sucrose(Sweet)

G

0.03%(w/v)

Caffeine (Bitter)

H

Ballygowan still water

J

0.15%(w/v)

NaCl (Salty)

K

0.04%(w/v)

Citric acid monohydrate (Sour)

(Table 2.6.1)

The candidates were told that the samples contained natural sweet, salty, sour and bitter compounds. They were asked to taste each sample individually and indicate whether the sample was sweet, salty, sour or bitter in the provided spaces. If the sample tasted like water they were asked to mark with a zero (0), if they were unsure of the taste they were asked to mark with a question mark (?). When tasting the sample the candidates were asked to swirl the solution around your mouth ensuring it contacts all parts of the tongue.

Between samples, candidates were asked to eat a piece of un-salted cracker and rinse their mouths thoroughly with still mineral water. A cut off point was set at 6 correct answers, 1 of which must be bitter or 5 correct answers, 2 of which must be bitter. Out of the 17 candidates screened, 7 were selected for bitterness training.

For the bitterness training candidates were asked to assign bitterness scores to unknown solutions based on a 0-100% scale, where a 100% bitter solution was taken to have a bitterness value equivalent to 1 g caffeine/L. Still mineral water was used as the 0% bitterness standard. All 7 displayed a strong ability to detect different levels of bitterness.

A range finding sensory study was performed to determine the optimum hydrolysate concentration for sensory studies. This was to ensure that the sample bitterness fell within the bitterness range of 0-100% where as stated above 100% bitter solution was taken to have a bitterness score equivalent to 1g caffeine/L and still mineral water was used as the 0% solution. The 15min hydrolysate sample from the 5% set of NaCN hydrolysate, with a DH of 12.05% was used in this sensory test. This sample was used as it had a slightly higher DH than the samples of interest, i.e. if they could accurately score the bitterness of this sample below 100 then they should be able to accurately score the bitterness of the samples of interest below the threshold of 100. The concentrations tested were 0.225% (w/v), 0.3% (w/v) and 0.45% (w/v). The optimum concentration was determined to be 0.45% (w/v).

Finally the hydrolysate samples at a concentration of 0.45% (w/v) were randomly presented over three days to members of the bitterness evaluation panel. At each sitting, panellists were firstly presented with solutions of 0.00, 0.25, 0.50, 0.75 and 1.00 g caffeine/l, which had been labelled as 0, 25, 50, 75 and 100 respectively. Panellists then assigned bitterness scores to the test hydrolysates on the basis of the caffeine standards they had tasted. Between samples, panellists were asked to eat a piece of un-salted cracker and rinse their mouths thoroughly with still mineral water. Figure 2.6.2 is a photo of the setup for the sensory evaluation.

(Figure 2.6.2)

2.7 Statistical analysis

Statistical analysis of the bitterness scores was carried out using the statistical programme R version 2.10.1©, One-way analysis of variance (ANOVA) and independent-samples t-tests were performed on sensory data. A significant result was defined as P < 0.05, a highly significant result was defined as P < 0.001.

Chapter 3

Results and Discussion

3 Results

3.1 TNBS Degree of Hydrolysis Curve

(Figure 3.1.1)

Figure 3.1.1 shows the degree of hydrolysis (DH) curves for each of the 5%, 10%, 15% and 20% protein concentrations. It would be expected that the curves would appear in the order of 5%, 10%, 15% and 20% but, unusually the sequence manifests in the order of 5%, 15%, 10% and 20%. Also it is seen that there is very little difference between the 10% and 20% in relation to their DH values. This result was unanticipated and so in order to rule out the possibility of any human or mechanical error causing this result, the 10% hydrolysis was repeated and again the same results were obtained.

3.2 Basic Gradient RP-HPLC

(Figure 3.2.1) need to edit on daras comp

Figure 3.2.1 shows the reverse-phase high performance liquid chromatography of the four selected hydrolysate samples with similar DH values but different protein concentrations. The DH values range from 11.37% to 11.68%. The gradient used for this set of graphs was the basic gradient. From the chromatograms it is evident that several peaks decrease with increasing protein concentration, similar results were obtain by (Spellman et al 2004) in relation to whey protein hydrolysates. These peaks have the approximate elution times of 28mins, 31mnis, 34mins, 39mins and 46mins. However, for some of these peaks such as that which appears at 39mins, a 'shoulder' has developed from chromatogram to chromatogram. The reduction of the gradient slope to improve resolution is only achieved at the expense of time. However, adjusting the gradient slope is important in optimising resolution of the peptides and proteins. To rectify this problem several test RP-HPLC chromatograms were carried out on highly hydrolysed casein samples to elect an optimum gradient which would pull apart these shoulders and give a clearer picture of what changes are occurring in the peak areas as we go from the 5% to the 20% samples (see appendix for chromatograms). Figure 3.3.1 represents the chosen extended gradient RP-HPLC. The peak which appears at approximately 41mins, 75mins and a block of peaks with elution times between 60mins and 70mins appear to decrease constantly from the 5% to the 20% protein concentration. The peak which appears at approximately 80mins however shows a slight decrease from 5% to 10% only to increase again when going from 10% to 15%. Thought there is apparent physiochemical differences between the 5% and 20% hydrolysate samples, RP-HPLC on its own is only an indirect indicator of bitterness and must be coupled with sensory studies to determine if there is actually a significant change in bitterness between the samples.

5% (w/v)

20% (w/v)

15% (w/v)

10% (w/v)

Retention Time

Detector Response @214nm

Detector Response @214nm

Retention Time

Retention Time

Detector Response @214nm

Retention Time

Detector Response @214nm3.3 Modified Gradient RP- (Figure 3.3.1)

3.4 Sensory

Given the relationship between bitter taste in protein hydrolysates and the presence of hydrophobic peptides (Ref) the sensory analysis is a key factor which ties all the results together. The hydrolysates generated at different protein concentrations but possessing very similar DH ranging from 11.37% - 11.68% were presented to a sensory panel trained to quantify bitterness. The sensory analysis was carried out over three sessions to achieve a more accurate result. A linear relationship was not observed between hydrolysate bitterness and the protein concentration at which the hydrolysate was generated (Figure 3.4.1). There is however a dip which appears at the 10% sample which may be of significance.

(Figure 3.4.1)

Statistical analysis of the sensory data was carried out using the R version 2.10.1©, to compare the means and to investigate if there is a significant difference between mean bitterness scores.

Table 3.4.2 ; ANOVA (Analysis of Variance) between the means bitterness scores of the samples. A significant result was defined as P < 0.05, a highly significant result was defined as P < 0.001.

 

Df

Sum Sq

Mean Sq

F value

Pr(>F)

Treatments

3

1283.85

427.85

1.5052

0.2184

Residuals

92

26252.2

284.25

 

 

ANOVA analysis demonstrates if there is any significant variance between the means when taking all four of the hydrolsates mean bitterness scores into account, as we can see the p-value is greater than 0.05 and therefore there is no significant difference between the means.

Independent t-tests are used to evaluate if there is a significant difference between two means. The below table 3.4.3 shows the resulting p-values, it is observed that the only significant difference is between the 10% and 15% hydroslate samples.

Table 3.4.3 ; Independent t-test between the means bitterness scores of two samples. A significant result was defined as P < 0.05, a highly significant result was defined as P < 0.001.

Between

p-value

Significance

5% & 10%

0.1967

Not significant

5% & 15%

0.7305

Not significant

5% & 20%

0.958

Not significant

10% & 15%

0.04418

Significant

10% & 20%

0.1

Not significant

15% & 20%

0.7253

Not significant

There is a possibility that enzyme activity is distorted above at high protein concentrations. There are several papers supporting this possibility such as that by (Ng, 1979) which discussed the effect of viscosity on subtilisin, (Spellman, 2004) where the effect of total solids on enzyme action was demonstrated. Findings by (Dang, 1998) are similar to that of (Ng, 1979).

To investigate this further two more sets of hydrolysis were carried out at 7.5% and 12.5% protein concentration and their degree of hydrolysis was quantified using the TNBS method.

3.5 Generation of hydrolysates at 7.5% and 12.5% & %DH

(Figure 3.5.1)

Figure 3.5.1 shows the first 60mins of the DH curves for each set of hydrolysis generated at different protein concentrations. The sequence of the curves appears to be 5%, 12.5%, 7.5%, 15%, 10% and 20%. A paper by (Tan, 2010) discusses the possibility that within an aqueous sodium caseinate solution of total solids concentration of above 10% protein - protein reactions occur and cause the solution to transition from being viscous-dominant to being elastic-dominant.

(Figure 3.5.2)

This information suggests that below 10% total solids concentration the curves behave as would be expected with the curve with the lowest protein concentration proceeding at the fastest rate of hydrolysis. Figure 3.5.2 shows the DH curves for only the 5%, 7.5% and 10% set of hydrolysis, which do appear in order i.e. 5% > 7.5% > 10%.

in the solution without any protein- protein interactions.

Figure 3.5.3 DH Values (TNBS) for prolyve hydrolysates at different protein concentrations ranging from 5% (w/v) to 20% (w/v) as a function of time.

Figure 3.5.3 shows the DH curves for only the 12.5%, 15% and 20% set of hydrolysis which also appears in order. Taking this into account the possibility that the enzyme behaves differently above and below 10% (w/v) is probable. Below 10% (w/v) the DH curves appear in order, with the lowest protein concentration proceeding at the fastest rate of hydrolysis, but the DH curves also appear in order above 10% (w/v). The paper by Tan, 2010 suggests that above a total solids concentration of ~10% (w/v) protein-protein interactions occur and forms a permanent elastic-dominant solution after an extended period of time without the application of a shear force to the solution. The solutions used for the hydrolysis in this study were prepared the night previous and then stored overnight at 4°C. This may have facilitated the protein- protein interactions referred to by Tan et al. Dang, 1998 has also put forward the possibility of protein - protein interactions at higher protein concentrations.

Conclusion

The sodium caseinate hydrolysates were generated at different protein concentrations and underwent physicochemical characterisation & sensory evaluation. From this it was seen that there was no significant difference in the bitterness of the hydrolysis samples with the exception of the 10% (w/v) hydrolysate (mean bitterness score = ) which was confirmed to be significantly less bitter than the 15%(w/v) hydrolysate (mean bitterness score = ) by an independent t-test. It would be expected that the hydrolysis would proceed at a faster rate for the lower protein concentrations and that the rate of hydrolysis would gradually decrease with increasing protein concentrations. However, this is not the case as seen in figure 3.5.1 (DH Values (TNBS) for prolyve hydrolysates at different protein concentrations ranging from 5% (w/v) to 20% (w/v) as a function of time). On the other hand when separated onto two graphs one for above and one for below 10% (w/v) the curves appear as expected. A paper by Tan, 2010 suggests that above a total solids concentration of ~10% (w/v) that the there is increased protein - protein interactions which may become permanent after 24hrs, protein - protein cross linkages at high concentrations is also suggested by Dang 1998 who examined the effect of viscosity on enzyme action. Though protein - protein cross linkages is an attractive explanation for the obtained results, it could not be verified as there was only a limited amount of time to perform this study.

Limitations of work

The main limitation in this study was time, there was only sufficient time evaluate the original 4 hydrolysis samples in the sensory studies. This is very limiting as sensory studies rely on human perception of a taste and their mental and physical state on at the time of the sensory evaluation, if an individual on the panel was tired their perception of the taste could be distorted and this would affect the overall bitterness scores. For this reason more samples would be needed to verify that there is a difference in bitterness and that the 10% hydrolysate sample is not an outlier or a result of human error.

Future work

Further work is needed in this area to determine if there is in fact a change in bitterness around 10% (w/v) protein concentration. Hydrolysis's set at closer protein concentration intervals would need to be generated to properly map any possible changes in bitterness.