Protein Hydrolysates And Removal Of Bittering Principles

Production And Uses Of Protein Hydrolysates And Removal Of Bittering Principles In It

Production And Uses Of Protein Hydrolysates And Removal Of Bittering Principles In It





A series of commercially prepared casein and soy hydrolysates and also an acid hydrolysed casein hydrolysate are presented below

Enzymatic method: a combination enzyme treatment of casein using papain for 18hr followed by the addition of a homogenate of swine kidney cortex, produced a hydrolysate with reduced bitterness.

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Using papain for 18 hr incubation

Add homogenate of swine kidney cortex

Flow diagram for the production of protein hydrolysate with use of enzyme papain source: clegg and Mc Millan (1974) Dietry enzymic hydrolysat of protein with reduced bitterness J. Food Technol 9:21

Treatment with activated carbon:

To a 100ml sample of hydrolysate prepared from 6.25% casein solution venous amount of activated carbon (0.210 1.0g g-1 protein) were added. The suspension was stirred for 60 min at 25c, filtered, and the extent of bitterness evaluated by a taste panel.

39 liters of 6.41%

solution, pH 8.3


59 litre –1 incubation (60oc, 4hr)



Centrifugation fitter press filtration

Incubation (25oc 1hr)

Centrifugation, fitter, press filtration

Pasteurization, lenzyme pasteurization enzyme mactivation) 83 – 85oc, 3min inactivation 83 – 85c, 3min

Cooling Cooling

Spray drying Spray drying





Protein 2. 94

Soya bean oil 2. 80

Starch 4. 48

Vitamin mixture 0. 50

Mineral mixture 1. 00

Water 88. 36

Preparation of casein hydrolysate

Casein was solubilized in distilled water by pH adjustment to 7.5 using 0.5N NoaH. 5% Na caseinate solution was hydrolysed by 1000 ppm pronase at 50oc until protein coagulation at pH 4.6 was undetectable.

Effect of enzymatic hydrolysis on some functional properties of wey protein.

As a liquid by – product of the manufacture of change and casein, whey has been considered to be a process affluent.

Source of proteins: Commercial concentrate of whey protein prepared by get filtration was obtained. Casein and dialysis whey protein concentrate were prepared by precipitation of casein from skin milk with Hcl at a Hp of 4.6. The precipitated casein was washed three times with water, resolubilized in water by adjusting the pH to 7.5 with NaoH, reprecipitated, washed and freeze dried. Dialysate whey protein concentrate was prepared by dialyzing the whey (pH 4.6) at 2oc for 72 hr against an excess of distilled water. The dialysate was then freeze dried, Grate A, low- heat, nonfat dry milk (NFDM) was obtained.

Sources of enzymes used

The enzymes used were obtained from the following sources: pronase, cal biochen; pepsin (1:10, 000) Cudahy; and prolase (EN – 21)


Gel filtration: Gel filtration studies were performed utilizing a Pharmacia K 15/90 column packed with G-50 Septhadex A 0.25 ml sample of a 2.5% whey protein so enzymatically hydrolyzed whey protein was chromatographed with 0.1m ammonium acetate buffer sol at pH 7.2 at a flow rate of 280 nm. The samples were adjusted to pH 7.2 before application.

Digestion procedure: suspensions of whey protein (2.5% w/w) prepared by gel filtration were adjusted with Hcl or NaoH to pH 2.0 for prepsin and 7.5 for pronase and prolase EB –21 and were held in a water bath at 50oc for 15 min before the addition of enzyme at a ration of 1 part enzyme to 75 parts protein the solution was incubated at 50oc and samples taken at selected time intervals. The digestion vessels were covered with watch-glasses. During the course of digestion, pH changed very slightly, i.e. x +0.3 unit the solutions were not buffered because of the influence buffer salt might have on foaming or emulsifying properties. The extent of proteolysis was monitored by formal titration A 5.0 ml aliquot of the hydrolysed protein solution was diluted to 15.0 ml, titrated to pH 8.5 with 0.02H NaoH, 2ml of neutralized for maldehyde added and again titrated to pH 8.5.


Whipping properties: The whippability characteristics of the protein sol were obtained by diluting an appropriate quantity of protein concentration of 4% protein. The pH of the sol was adjusted, heated rapidly and immediately whipped at speed 8 in a kitchen Aid, Mode 3-c mixer equipoled with a wire whip. Specific volume was determined by carefully transferring the foam to a tarred 160 ml crystallization dish and weighing, the stability of the foam was determined by covering the weighed dish with a ½ in mesh screen. The foam was then inverted over a funnel and liquid draining from the foam was collected. The time required for collection of liquiel equal to ½ of the weight of the original foam was recorded. All measurements were made in triplicate.

The effect of enzymatic hydrolysis was-determined by adjusting a 4% sol to the optimum pH for the particular enzyme and held in a water bath at 50oc for 15 min before the addition of enzyme at a ratio of 1:75. after incubation for desired times 50 ml aliquots were heated rapidly to 85oc and immediately whipped.

Emulsion Capacity: The emulsifying capacity was determined by a procedure similar to that of Webb et al (1970). An aliquot of 2.5% protein sol was taken from a standarclized whey protein sol which was equivalent to 25 mg of protein. This was added to 100 ml of 1.0m NaCl solution is a 400 ml beaker at 25oc and the and the mixture tarecl. Refined corn oil was added from a 500 ml graduated cylinder which had been fitted with a stop cock at the base to facilitate delivery of the oil. The rate of addition was maintained constant at 1ml/sec during continuous stirring. Electrical resistance, monitored by a voltmeter was used to determine the break point of the emulsion. The emulsifying was calculated as the g of oil required to reach an infinite electrical resistance minus a blank, which considered of the g of oil required to reach an infinite electrical resistance of 100ml salt sol (51.2g) divided by the amount of protein (mg).


Washed three times with water


Depreciated and washed


Dialysate whey protein concentrate prepared

By dialyzing the whey (pH 4.6) at 2oc for 72hr against an excess of distled water.

Grade A, low-heat, nonfat dry milk (NFDM) was obtained.

A flow diagram for the production of nonfat dry milk (NFDM) using casein precipitate source: Morr. C.V, Swenson, P.E and Richter, R.L (1973). Functional characteristics of whey protein concentrates. J. food Sci 38: 324.

A table showing Emulsifying capacity of Casein whey protein concentrate from gel filtration, whey protein concentrated by dialysis and nonfat dry milk (NFDM).

I protein source & oil/mg protein

Casein 1. 88

Dialysed whey protein concentrate 2. 99

Sephadex-processed whey protein concentrate 2. 97

NFDM 2. 17

II whippability of whey protein concentrated by gel filtration

Variable specific vol foam stability

Ml/g (min)

i. Temp of heating (4% sol)

oC; no hold pH 8.5

25 8. 3 0

35 10. 4 35

45 11. 4 66

55 11. 6 72

65 11. 9 74

75 12. 2 75

85 14. 0 77

95 14. 3 80

75 (held for 5 min) 14. 5 79


ii protein content (w/v)

85oc, pH 8.5

2% 12. 4 65

3% 13. 7 83

4% 14. 0 94

5% 14. 7 102

6% 15. 5 110

iii whipping time (min) (4% sol

heated to 85oc) pH 8.5

2 13. 3 60

4 13. 7 73

6 14. 3 74

8 15. 6 98

10 17. 4 126

iv pH (4% sol heated to 85oc)

2 13. 8 29

3 13. 9 28

4 12. 5 80

5 11. 4 77

6 11. 3 62

7 13. 2 85

8 13. 8 101

9 14. 0 107

8.5 (Ca (OH) 12. 9 83


v Added CMC w/v (4%

sol heated to 859 pH 6.5)

0. 0 11. 4 71

0. 1% 11. 0 166

0. 2% 12. 4 205

0. 3% 12. 9 295

0. 4% 13. 2 360

0. 5% 13. 3 500


vi Miscellanceous

Egg whites 9. 4-95 130 min

Casein 14% sol heated 55% pH85 11. 6 25 min

Using a 6 min whipping time except group 111, where time was varied


Food product applications: The flavour of the debittered hydrolysates has been found to be compatible in all foods or beverages, where a beezy borty character is desirable Addition of debittered hydrolysate to Tomato or Onion based soups, sauces, drinks, or extruded products were ranked most acceptable at the 1-3% w/v level. The debittered product has also been compounded into tablet or water form with various flavoring additives, Likewise, the addition of these debittered 3hydrolysate to bland soy based proteins foods or two various cheese products has proven to be desirable.

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The nutritive qualities of casein hydrolysate which were untreated and carbon-treated, and carbon-treated hydrolysates supplemented with amino acids are shown in the table below. The PER result show that untreated hydrolysate had a nutritive value (3.38) similar to that of casein (3.68)

On the other hand, activated-carbon-treated hydrolysate has a very low PER values (0.55). Supplementation of the treated hydrolysate with trytophan, phemylalanine and arginine resulted in a marked increase in the PER of the diet o a level (3.03) which was not different from that found for the untreated hydrolysate clearly the two most deficient amino acids in the carbon treaded hydrloysate were tryptophan and phenylalanine, apparently due to the selective adsorption of these amino acids, or of peptides containing them by the activated carbon.

A TABLE showing the nutritional quality of casein hydrolysate supplemented with several amino acids supplementation with amino acids mg 100 g-1 diet


Protein source Try Phe Ary Glu Met Per

Casein control

Casein hydrolysate untreated

Casein hydrolysate treated

Casein hydrolysate treated

Casein hydrolysate treated

Casein hydrolysate treated

Casein hydrolysate treated

Casein hydrolysate treated –




70 –




240 –





– 788






473 100







100 3.68 At 0.18

3.38 Aot 0.13

0.55Bt 0.19

0.24Bt 0.20

1.95ct 0.15

3.03Det 0.12

1.53ct 0.15

2.78Et 0.09


Further nutiritive advantages: According to Murray and baker (1952) A treatment of enzymic casein hydrolysates with activated carbon resulted in a substantial improvement in the taste of preparations.

A debittered protein hydrolysate serves as preservation of reasonable nutritive value. However, the bitter principles are retained for nutrition and medication. During the hydrolysis of protein, the toxicity of the compound are reduced.


The bittering factors: One of the limitations of protein hydrolysate is the bittering principles in it.

The bitterness sometimes is produced in fermented products therefore decreasing their qualities. The bitterness is produced by bitter partials and their derivatives formal during ageing process of these fermented products.

The bitterness is also produced by enzymatic hydrolysis of protein very often to decrease the value of the products.

Another important key for the bitterness is the hydrophobic of a peptide.

The amino group is also a bitterness producing functions.

The enzymic digestion of protein such as casein coprecipitate and soy protein invariable leads to the formation of bitter tasting peptides. Ths defect limits the acceptance of the hydrolysates for incorporation into foods. Caffeine is also a bittering principle in food system such as beverages.


The unpalatability of tese hydroolysate arises mainly from the formation of bitter peptide and amino acids liberated during the hydrolytic process, Eriksen and fagerson (1976).

The bittering principles limits the acceptance of the hydrolysates for incorporation into foods. The bitter peptide which arises during the hydrolystic process decreases the value of the product.


The adsorption methods of debittering skim milk hydrolysates for incorporation into beverages preliminary experiments using hydrolysed commercial casein indicated that the resulting bitter peptides were mostly hydrophobic as they were almost completely eliminated by hydrophobic affinity chromatography on hexycopozy and octylepoxy scpharoses. Butyl epoxy and alkyl amino sepharoses were less effective in debittering.

Although B cyclodextrin a Suzuki (1975) claimed was effective in masking the bitterness of casein hydrolysates, further experiments are needed for establish its safety as a food additive. Presumably, the hydrophobic inner cove in the cyclodextrin structure extent some attraction for the hydrophobic bitter peplides. Activated carbon adsorbed bitter peptides from casein hydrolysates Marray and Baker (1952) puplished the first report on the use of activated varbon for reducing the bitterness of protein hydrolysates. Stirring pronase-hydrolysed casein with 200% carbon (Witco 48 x 105) vs. protein for 2 hr at room temperature reduced the bitter taste of the hydrolysate on an acceptable level.

The results of these casein hydrolysate experiments suggests that activated carbon treatment is a promising techinique for improving the palatability of hydrolyzed skim milk.

Dibettering of skim milk hydrolysates: As a first step, protein in reconstituted skim milk was hydrolysed with minimal coagulation. This was accomplished by adding 1.0-4.0% enzyme vs protein after pH adjustment to the enzyme optimum and then inclubating at 5oc for 16-24 hr. pronase, ficin, papain, trysim, and protease from A. oryzae and B. subtilies. Yielded acid-non coagulable hydrolysate but bromelain and pepsin did not. As trysin, pancreatin, and a. oryzae protease imparted objectionable flavours to the hydrolysates, hydrolysis was carried out using 4% ficin or 0.5% pronase vs protein at 5oc for 20-24hr. The next step was a screening study of hydrophobic adsorbents and plastics for their capacity to remove bitter peptides from milk hydrolysates sephadex LH-20 and phenoxyacetyl cellulose decreased the bitterness of pronase and ficin-hydrolysed skim milk to approximately 50%. This finding confirmed the hydrophobic charater of the bitter peptides. The inorganic adsorbents Bas 04 and fuller’s earth exhibited negligibe bitter peptidedrption capacity as did the plastics low-density polyethelene and polyvinulidene chloride.

Anion exchange treatment of skim milk hydrolysates

As the treated hydrolysates were salty, they were passed through a weakly basic anion exchange resin, Amberlite 1R-45, successively regenerated with NaoH and gluconic acid to replace diloride with gluconate Helbig et al., (1978)


10% SNF skim milk powder 100ml

Add 0,14g ficin or 0.02g pronase and incubate at 5oc for 16hr, pasteurize

Protein-hydrolysed milk or

Pass through a glass fibre column shake with 3.6g Darco kB

Class-treated milk hydrolysate carbon for 2hr

Shake with 1.0g Darco kB carbon centrifuye or filter

For 2h centrfuge or filter

Carbon treated milk hydolysate

Pass through an Amberlite

1R-45 column

chloride – removed milk hydrolysate


powdered debittered protein hydrolysed milk.

Prcessing flow sheet for dibittered protein hydrolysed milk. Source cleg9, and McMillan (1974) J. Food Technol 9:21




The absorptive effect of the phenol-formaldeliyde resin (Duolite 5-76) on aromatic amion acids and peptides present in the protein hydrolysates is illustrated in fig 1 and 2.







Fraction, L

Figure I

Hydrophobic chromatography pattern of N – Z Soy enzymic digest of Soy protein.











Figure 2

Hydrophobic chromatography pattern of N – X Amine A casein hydroysate on duolite – 761 resin.


As can be observed in fig 2 the shape of the total solid and conductivity curves follow normal haussian-shaped distributions with peak concentrations occurring in fraction 5. however the 280 nm absorption carve has its concentration peak appearing in fraction 6 and shows positive skewness. A similar retardation of the 280 nm absorption peak was consistently found in other hydrolysate fractionations and was also observed for the larger scale fractionation of B-2 soy protein hydrolysate (fig 1) in this instance, the total sliods/conductivity curves show peak value after 60L of hydrolysate have been eluted, followed by the 280nm absorption values which peaks after 70L of processed material have been eluted.

In order to gain further insight into the hydrophobic binding interactions occurring between the phenolic structured resin and the mixed distribution of aromatic and heterocyclic peptides and anion acids in the hydrolysates, various anion acid analysis of individual and pooled fractions of no bitter and bitter materials were undertaken.




In other to develop a practical debittering method for anion acids and peptides several debittering methods were studied. It was found that looking acidic anion acids to and acetylating of bitter anion acids is very effective to remove the bitterness from their concentrated solution. For debittering by mixing with additives, skin milk and other peplide compounds were effective the bitterness. Gelalimzed starch was found to useful for debittered because it takes bitter substances into it’s not structure.

It is widely known that bitterness sometimes is produced in sake “miso nalto” and other fermented products, so decreasing their qualities. This bitterness is produced by bitter peptides and their derivatives formed during aging process of these fermented products. Enzymatic hydroysis of proteins also produces bitter peptides very often to decrease the value of the products.

Bitter peptides has been treated with carboxyl peptidases to reduce the bitterness by some research groups.

Extraction with alcohol or treating on resin has also been attempted to remove the bitterness from peptides.

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All these debittering methods involved removing the bitter principles by extraction or absorption or by modifying the structure of the bitter peptides.



a. Debittering by modifying the structure of bitter anion acids.

The mechanism for the bitterness of anion acids and peptides is clear cause the simplest way to remove the bitterness is to change the structure of the bitter anion acids and peplides. The effect of debiltering by changing the bitter structure produces a sour taste with various compounds used as salt free derivatives, and also produces a salty and Umami taste as sodium salts. The anion groups of this compound were blocked by an acetyl, aspartyl or glut amyl residue.

b. Debittering by adding substances for effective masking of the bitterness

i. Debittering by & cyclodextrin or starch shows that 1.5 requirement of & cylodextrin was necessary in order in order to mask the bitterness of amino acids, Here & cyclodextrin is presumed to grap the hydrophobic functions of bitter amino acids to decrease their bitterness, therefore 1.5 equivalent of & cyclodextrin could be estimated as effective for the peptides whose bitterness potency is stronger than that of amino acids. However, 1.5 equivalent of & cyclodextin was not wrapping ability of & cyclodextrin seems not to be as high as possible. Reason based on the probality that the peptide molecules are too big for & cyclodextrin. Another reason might be that the peptides posses more than two hydrophobic function which & cyclodextrin has to wrap. The fact that a large excess of & cyclodextrin was necessary to reduce the bitterness of the peptides explains these phenomena. Starch was expected to reduce the bitterness of the samples because starch has a net structure, which is suppose to cover bitter compounds.

ii. Debittering by skim milk protein and casein by drolysate. Here the peptide compounds are expected to decrease the bitterness, because there should be some affinity between the proteins and anino acids or peptides. The protein and protein hydrolysate were effective to decrease the bitterness as expected, because the result of a sensory, analysis taken shows a successful debittering by skim milk, soybean casein, whey protein concentrate and casein hydrolysate. He these materials are peptide compounds.’

iii. Debittering by fatty substances. Hydrophobicity of the molecule is one important factor for bitterness. Fatly substances might be effective to reduce the bitterness because of their affinity to hydrophobic compounds. A sensory analysis of a mixture of bitter substances and creaming powder margarine and vegetable oil is carried out and the result however shows that fatly substance were not very effective with the exception of creaming powder. The creaming powder probably decreased the bitterness by foaming an emulsion to wrap the bitter amino acids or peptides. The mixture of margarine and vegetable oil in water were easily separated into two layers, which was probably the reason why then did not work effectively.

iv. Debittering by acidic amino acids. Studies were finally done on debittering by acidic amino acids, aspartic acid, glutamic acids, and taurine. Area et al reported that acidic peptides reduces the bitterness of casein hydrolysates by try sin and other bitter substances. Acidic amino acids should have the same function a acidic peptides which worked to reduce the bitterness. Taurine of course is not an acidic amino acid, though it has a sulfonyl group and the solution shift to the acidic region. An acidic solution of taurine might be as effective for debittering as asp artic acidic or glutamic acid. The result of the sensory analysis carried out shows that all the amino acids instead of the bitterness. Turine was the only amino acid which did not produce sourness although it reduced the bitterness as well as other acidic amino acids.

Debittering the bitter peptides from tryptic hydrolysates of casein copercipitate by immunoadsorbent Affinity chromatography.

Specific antibodies were prepared against a bitter peptide, which had been previously isolated from tryptic hydrolysates of casein co-precipitate the antibodies were immobilized by covalent attachment to sepharose 4B and the resulting immunoadsorbents were able to remove bitterness from these hydrolysate. However, the was not possible to completely remove absorbed bitter peptide from the immunoadsorbents even when using strongly defoaming solvents..

The enzymic digestion of proteins suds as casein, casein corpreciptitate and soy protein invariably beads to the formation of bitter tasting peptides. This defect limits the acceptance of the hydrolysates for incorporation into foods.

Immunoadsorbent affinity chromatography is used in research for the specific purification of antibodies and antigens. However, this technique might be employed commercially to slectively remove undisirable flarour components from foods by means of immobilized antibodies against these component. Before the use of immunodsorbent affinity chromatography to remove undesirable consitiuents from food products can be considered for commercial application, the riversiblity of the processneed to be demonstrated.

Normally the dissociation of antigen-antibody complexes can be achieved dimply by adjustment to low pH or by the use of unfolding agents or chactropic ions. However, even strongly chactropic agents such as 5m k1 0. 4m NH4Scn did not completely dissociate the BP-BP antibody complex. Such strong binding of small peptide to it’s antibody was unexpected and the reasons for this are unclear.

Methods of debittering: 1. coupling of bitter peptide (BP) to thyroglobulin carrier. Because of its small size (M> W> 1500) it was necessary to couple the peptide to a large protein in order to elicit antibody response, and bovine thyroglobulin was chose for this purpose.

Initially the BP was coupled to thyroglobulin using CMCS by the method previously decribed for the binding of biotin to serum albumin in this preparaion 10 moles BP were bound per mole of htyroglobulin, as determined by amino acid analysis using the a LC method of pearce. When the amino groups of BP were protected prior to CMCS coupling the binding was increased to 18 moles BP/mole thyrolylobulin, 8mg BP was dissolved in 5ml water, 10ml pyridine and 11mg succinic anlydride were added and the solution was stirred for 30 min at room, temperature. The solution use then concentrated to 3ml with a rotary evaporator. The treated bite peptide was coupled to throglobulin as before, purified on sephadex 925 and greeze-dried. The preparation was uses for the immunizations.

Preparation of antiserum against BP: 20mg of BP-throglobulim conjugate in 10ml physiological saline was emulsified in 10ml freund’s complete adjuvant. Two rabbits were each given five intramuscular injections of 2ml containing 2mg conjugate. These procedure was repeated five weeks later followed by three more immunizations 11, 14 and 20 weeks after the first injections using the alum adjuvant instead of freund’s adjuvant. Beginning after the second immunizations the rabbits were bled by earvein puncture about two weeks after each treatment. The blood was allowed to clot and sera were separated from the cloths by centfrifugation and assayed for bitter peptide binding capacity.

Redioimmunoassay: BP was trace radio-iodinated with Na 125 1 and lactoperoxidase according to the method of Marchalous. The specific activity of the labeled BP (BP125 I) was about 7000 CPS/N mole BP125 I increasing quantities of BP125 I were incubated for 30 min at 37oc in tubes containing 25 N/ serum diluted to 5500 N/ with distled water. The incubated solution were then applied to 10ml columns of ultro gel ACA 4 and eluted using 0.01m sodium phosphate in 0.15m Nacl pH 7.4 (PBS). The radioactivities of the void volume peaks we determined by a packard gamma scintillation spectrometer until saturation was reached. The amounts of Bp125 I needed to saturate the sera were noted.

Non-specific binding was determined by incubating normal rabbit serum with Bp1251. also anti serum from the second bleeding was incubated with a radiodinated peptide similar to BP (specific activity about 8000 GPS/Pmole peptide). This peplide has been isolated by gel filtration on sephadex G25, followed by ion-exchange chromatography on DEAE cellulose from a tryptic hydrolysate of casein and comprises segment 47 or 63 of that protein. It contains three tyrosine residues and a number of the other amino acids present in BP.

Adsorption of bitter peptide. The ability of these three preparations to absorb BP was determined by the passage of solutions of BP125i, non labbled Bp and extremely bitter tryptic digest of 4% coprelipitate through the columns at 5oc from bitterness comparisons with BP solutions of known concentrations this hydrolysate appears to contain approximately 0.7mg BP per ml. The eluates were assayed for radioactivity or N content or, in the case of digest, were tasted. Also, corprecipitated digest was passed through a column containing preparation 2 with the addition of propend or urea to the digest and to the PBNS eluant in order to observe the effect of these protein unfolding agents on the binding capacity for BP and ease of subsequent dissociation of the immune complex.

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Improvement of product Attributes of Mayonnaise by Enzymic hydrilysis of Egg Yolk with phospholipase A2 Egg Yolk with phospholipase A2 Egg yolk fermented with pancreatic phospholipase A2 has been shown to be a more potent emilsifier for mayonnaise than untreated egg yolk. The mayonnaise withstood heating at 100oc for 30 min without the emulsion breaking. The treatment also affected a considerable thickening of the product. Animal feeding trials with lysolecithin and fermented egg yold suggested that the enzymic hydrolysis does not pose any doxological hazards.

Mayonnaise can be considered as an acidic oil- in–water emulsion which is stabilized by egg yolk. The stabilizing power of egg yolk is due mainly to the presenbe of Lipoproteins (lipovitellin, Liporitellin) which constitute 30% of the yolk solids. Food regulations in holand specify that the oil content of mayonnaise must be about 80% and the pure egg yolk content must be 6%. Other sponable emulsified dressing containing loss oil and egg yolk are usually thickened by the addition of starches or other thickening agents.

On of the problems in mayonnaise production is the problems in mayonnaise production is the breaking of the emulsion, which leads to oil exudation. This occurs when the temperature is raised above 70oc, or cooled below 0oc, or when too much shear is applied. One of the consequences of this instability is that the product cannot be pasteurized.

Some years ago, Van Dam (1978) discovered that enzymic treatment of egg yolk with phospholipase A2 (E.C.3.11.4) considerably improves the stability of the emulsion to heat treatment.

Further more, this treatment results in a remarkable thickening of the product, which permits a reduction in the amount of thickening agents needed in spoonable emulsified dressings.

Treatment of egg yolk with phoslipase A2 results in hydrolysis of the phospholipids –(Lecithin) into their corresponding lysophospholipids.

The effect of extracted lecithin or some phosphelogical parametere has been studied because of the relatively low phospholipid content of egg yolk – (2004g kg –1 of solids). Rat feeding trials have been conducted to compare enzymically hydrolyzed – Soya bean lecithin (Bolec z) and to evaluate hydrolyzed egg yolk for any biological effects.



Enzymic Modification: phospholipids A2 was obtained from porcine pancreas. This enzyme is present in the acetone defatted pancreatic powder but require some further purification for reasons of taster. The degree of conversion of egg yolk phospholipids depends on the amount of enzyme added, the reaction temperature, the incubation time and the salt content of the egg yolk. The calcium concentration of egg yolk is deficient to give an optimal reaction rate for phospholipase A2, which needs Ca 2+ ions for activity. This readion was found to be independent of the pH between values pH 6 and 8 the optimal condition conditions were determined for the modification of egg yolk. The conversion of phospholipides was measured according to a method described by Dole which was slightly modified. Exactly 3g egg yolk and 3ml water were mixed in a 75 ml centrifuge tube with 2g sand and 30 ml Dole reagent (isopropyl alcohol heptane 2 s04 ( 1 mol litre –1) 40:10:1). The mixture was shaken thoroughly and 18 ml heptane and 12ml water added. The mixture was centrifuged and 10 ml of the supernatant liquid was titrated with alcoholic NaoH (0.02 mol liter –1) using palmitic acid solution as a standed. The conversion was expressed as a percentage of the maximal conversion of all phospholipids.

H2C O – C – R1

R2 – C – O C H + H2O phosphoslipase A2



H2C – O – P – O – X


H2C – O – C – R1

HO C H + R2 – COOH

H2 C – O – P – O – X


Schematic representation of the enzymic hydrolysis of the ester bond at the C-2 position of phosphor glycosides by phospholipase A2. Croup X represents any of the naturally occurring residues which are found in phosphoglycerides.

Mayonnaise preparation: Mayonnaise was prepared in 2-kg batches. Water, Sugar, salt, vinegar, and egg yolk. (fermented or non-fermented) were emulsified using a kenwood mixer. After de – aeration under vacume and homogenization in a cocoid mill (presto mill) the product was stored in twist – off jars of 180g capacity.

The emulsion was sterbilized by the addition of potassium sorbate (2g kg –1). The stability of the mayonnaise was determined by heating the product in a closed jar at 100oc for 30 min products were visually appraised for oil exudation after cooling to room temperature, comparison of this method with centrifugation produces has shown that this procedure is sufficiently accurate to indicate whether oil or exudation occurs or not.







60– 60-

50 – 50-

40 – 40-

30 – 30-

20 – 20-

10 – 10-

0 10 20 30 40 50 60 0 10 20

Time (min) salt content of egg

Yolk (%)

(a) Conversion rate of egg yolk phospholipids a function of the amount of enzyme present. Temperature 55oc; pH 7.0; salt concentration 100g litre N,1000; 0,2500; A,5000 and o,10000 14 kg-1 egg yolk. (B) conversion rate of egg yolk phospholipids as a function of the salt concentration in the egg yolk

tempreture 55oc pH 7.0; 500 14kg –1 egg yolk fermented time 30 min.

Rheological characterization: In order to characterize the differences between mayonnaise containing fermented and non-fermented egg yolk. Samples were prepared with egg yolk fermented to different extents the characterization was performed by rheological techniques applying small deformations. So as not to disturb the structure the test.

Results; Heat stability: All mayonnaise sample prepared with fermented egg yolk which was for converted above 500% proved to be heat stable. At lower conversions, heat caused oil exudation. This stability allows the introduction of a pasteurization added preservatives or acid.

Thickening effect: Both fomentation and subsequent heating induced a thickening of the product. The shear storage modulus of mayonnaise containing normal egg yolk decreased with increasing tempreture, after the heating and cooling cycles the final modulus G was lower than the product before heating and the oil separated.



A major finding has been that reverse protease do occur under a suitable condition and are not just the result of hydrophobic physical aggregates. The reverse reaction resulted in a large reduction in perceived bitterness of model peptides. The technique may be applicable to monitoring changes during processing/maturation of a variety of foods.

Knowledge of the processes occurring during hydrolysate manufacture has been increased Two examples of discoveries with important implications are.

Reverse protease reactions may increase the amount of yield – reducing insoluble residue formed during hydrolysate production.

Transpeptidation reactions occurring during hydrolysis at high casein concentrations could make bitterness worse by rearranging peptides into more hydrophobic (and therefore bitter) specie.

The result of the debittering experiments are summarized in the experiment done in the practical debittering methods, it shows that each bitter substance needs a different debittering method. To remove the bitterness of high concentration solutions of hydrophobic acids, acytylation of the amino acids to hydroplicbic amino acids was effective, hooking acidic amino acids was also effective. For the debittering of peptides, peptide compound such as skim milk, soybean casein and so no were effective.

Gel atomized starch was effective for all the bitter compounds, and the recommendations for using gelatinized starch for debittering if no other effective anti bitter substances can be found was made of the variety of adsorbents tried, activated carbon and glass fiber were most practically feasible for debittering skim milk hydrolysate. The saltiness of the debittered product was dreaded by an amino exchange treatment. The product treated with cheese whey like appearance maintained good solubility in acids, flavour and nutritional quality. Two bitter peptides were isolated from the butanol extract of the activated carbon used in debittering pronase hydrolysed skim milk and partially purified by chromatography. The amino acids of Glu2, Pro3 val, lev1, Tyr4, phe1 and Glu4, pro2, tyr2, phe2 are compositions of the two fraction this saperated.

Pancreatic hydrolysis of Soya Lecithin’s or egg yolk does not result in any modification of the biological response when these materials are fed to rats in shorter studies.

Production And Uses Of Protein Hydrolysates And Removal Of Bittering Principles In It

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