الفهرس 
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Abstract
This work was carried out to determine the activity of proteolytic enzymes during the ripening of low-fat Ras cheese. As proteolysis plays
a major role in the development of texture and flavour in most rennet curd cheese varieties during ripening.
The study was divided into three parts:
Part I: Effect of fat content on the activity of starter bacteria.
This was monitored the changes occurred in the pH, acidity, and bacterial count of milk of different fat content which inoculated with starter culture at different intervals of incubation.
The result indicated that:
- pH, was slightly increased while the acidity decrease with the decrease in fat content.
- The total counts bacterial was decrease with the decrease in fat content in milk at different intervals of incubation.
Determination of starter bacterial count and quantity of rennet which were retained in the curd at pressing:
Total count bacterial retained in the curd after whey separation showed a pronounced decrease due to the reduction in fat content.
Effect of fat content on the rate of retention of rennet in cheese curd and whey, expressed as rennet coagulation time (RCT) per seconds. Results indicated that the rate of retention of rennet in the curd, was proportional with fat content in the curd, it is lower as the fat content in milk is higher. The average values of RCT for full-fat, reduced-fat and low-fat cheeses are 843 sec., 875 sec and 962 sec, respectively.
The opposite trend was observed for the rennet recovered in the whey. The RCT was greater for full-fat whey-cheese and lower for the reduced or low-fat whey cheeses.
Part II: Contribution of rennet, milk proteases and bacterial proteolytic enzymes to the primary proteolysis in cheese analogues of different fat content:
In this part of study the contribution of different proteolytic agents to primary proteolysis in different fat cheese analogues as well as changes in pH and the environmental conditions of cheese analogues were determined:
1- Changes in the pH of the analogues during keeping time pH values of the analogues on respective sampling days showed slight increase with keeping the analogues. Especially, the analogues with full-fat (1F, 2F and 3F) showed somewhat higher pH. After 30 days the analogues gave pH of: 5.47, 5.66 and 5.70 for the analogues: 1(1L, 1R and 1E), respectively while it was 5.22, 5.37 and 5.70 for the analogues 2, (2L, 2E and 2F) which containing plasmin, where is 5.04, 5.12 and 5.39 for the analogues 3, (3L, 3E and 3F) which containing plasmin plus starter bacteria.
2- Protein degradation of cheese analgoues:
2.1. Action of chaymosin and plasmin. Analogues made from paracaseinate containing active chymosin and plasmin with different ratio of fat were made (1L, 1R and 1F).
- Values of SN/TN increased at higher fat content analogues, and with their keeping time.
- The values of PTA-SN/TN had the same magnitude as that of WSN.
- The results demonstrated the increase in the production of AAN (Amino Acid Nitrogen) with keeping time and an enhanced rate of production was found at a higher fat content.
2.2. Action of milk proteinase (plasmin):
In all instances with keeping time and at higher ratio of fat cheese analogues gave higher values for all indices of protein proteolysis. At 30 days keeping time, the full-fat cheese analogues (2F) gave a remarkable increase in WSN/TN compared with the low-fat (2L) and reduced-fat (2R) cheeses. It was 22.92, 14.56 and 14.89, respectively. This rate of production of SN is reflected on the rate of production of PTA-SN. It was 3.12, 2.77 and 1.72% of the total nitrogen for the 2F, 2R and 2L cheese analogues respectively.
When AAN was taken in account, the low-fat and reduced-fat analogues represented 75.31 and 91.1% of AAN produced in full-fat analogues.
2.3. Action of milk proteinase plus starter enzymes:
Results on the effect of milk proteinase plus added starter on the rate of protein degradation showed the same magnitude as that for the action chymosin or milk proteinase. The WSN/TN, PTA-SN/TN and AAN values after 30 days were higher for full-fat (3F) compared with low or reduced-fat analogues. The WSN of low (3L) and reduced (3R) are 70.02 and 73.87% of the full-fat (3F) analogue, but the PTA-SN values are 47.5 and 67.62 and the AAN values are 57.09 and 62.09 of the full-fat respectively.
2.4. Urea-PAGE analysis:
The electrophotograms indicated the early degradation of -casein compared to the degradation of S1-CN. With progress in keeping time, the degradation of S1-CN progressively increased. However the results not clear to descriminate between cheese analogues on the basis of fat content. The results indicated that while both S1- and -caseins were hydrolysed, -caseins hydrolysed more extensively, resulting in the formation of -caseins.
Part III: The use of slurry in production of low-fat Ras cheese:
This part of study dealt with effect of fat reduction on the properties and the quality of the low-fat Ras cheese with or without slurry.
The study was designed to prepare milk samples containing different fat levels by mixing whole milk with skim milk to give levels of fat 1, 2, 3 and 4% fat, respectively. The cheese curd after whey drainage was divided into two portions; portion A treated as control, while portion S treated with slurry. The results indicated that:
The chemical composition of the resultant fresh cheese indicated that as the fat content in cheese decreased, the moisture, protein and pH were increased; while, the salt, residual rennet and total bacterial count were decreased. The results indicated that the increase in moisture content in cheeses with the added slurry, a decrease in the protein and an increase in salt content.
Addition of slurry increased either the percent of NaCl or the S/M in the low-fat cheeses.
Changes occurred in Ras cheese during ripening:
1- The moisture content of cheeses decreased with the progress in ripening period, the fat, protein and salt (NaCl) were increased with the progress in ripening period.
2- Comparing cheeses with added slurry (S1, S2, S3 and S4) with that without added slurry (A1, A2, A3 and A4), the cheese with added slurry kept higher moisture than the others up to the end of ripening period, but, it revealed higher content of fat and lower protein content than that without slurry during the extend of ripening period.
Evaluation of cheese quality:
1- The low-fat cheeses showed lower values of MNFS compared to the MNFS of high fat cheeses and this trend was continued up to the end of ripening period.
2- Addition of cheese slurry to Ras cheese curd has an increasing effect on the MNFS values which has an advantages property for the low-fat cheese.
3- The PDM represented about 44% in the cheeses of the higher fat milk (4% fat), it approximately represent more than 65% in the low-fat cheese (1% fat cheese milk) which may produced harder texture cheese and delay the ripening process.
4- Addition of cheese slurry increased the ratio of M/P at a given fat content, and might be expected to be a favourable factor enhancing the ripening properties of low-fat cheese.
5- The salt in moisture (S/M) level was proportionall directly with the increase in fat content in cheese. The higher the fat content cheeses were the higher in S/M. Although the S/M increased with the progress in ripening period as a result of the decrease in moisture, addition of slurry did not give an obvious trend but in general, cheeses with added slurry showed lower S/M values with the progress in ripening period.
6- The pH of low-fat cheeses was generally higher than that of high fat cheeses. With the ripening period the decrease in pH was approximately the same for all cheeses.
The pH decreased with added cheese slurry compared to that without slurry.
2.c. Ripening process:
The ripening process of cheese is very complex and involves microbiological and biochemical changes to the curd resulting in the flavour and texture characteristic of the particular variety. The resulting biochemical changes are determined by curd composition, microflora, residual coagulant and residual milk enzymes in the curd.
1. Ripening agents:
1.1. The activity of the residual rennet in cheese during ripening:
- It is clear that the fat content affected markedly the activity of the residual rennet in cheese. For fresh cheese curd, the rennet activity determined as, RCT, were 115, 112, 147 and 175 min for cheese made from 4%, 3%, 2% and 1% fat cheese milk ,respectively.
- During cheese ripening, an increase in rennet activity was observed. At the end of ripening period, the RCT values represented 47, 68, 76 and 66 of the RCT of the fresh cheese curds.
- The cheese slurry decreased the activity of the coagulant, specially with the higher fat content cheeses. At 1% fat, the RCT of the control cheese (A1) is equal to that of slurry treated (S1) cheeses, while at 4% fat the RCT of the slurry treated (S4) cheese was 15.65% higher than that of the control (A4). This phenomenon of the effect of cheese slurry on the residual rennet activity is not clear although the slurry have an improving effect on the compositional and bacterial count of cheese. This retarding effect of cheese slurry on the residual rennet activity was not only observed with fresh cheese, but more decrease was noticed with the progress in ripening period.
- At the end of ripening period the RCT of 1, 2 and 4% fat cheeses were 29.31, 13.49 and 22.55% higher than the RCT values in fresh cheese.
A.2. Bacterial count:
- Flavour development in cheeses requires lactic acid bacteria and enzymes.
- The bacterial count in cheeses increased with the increase of fat content.
- Addition of slurry increased the bacterial count at all instances and the bacterial count of low-fat cheeses improved markedly in the presence of slurry. At 1% fat cheeses the bacterial count was 176 and 156 x 106 for the added-and without-slurry cheeses respectively.
During cheese ripening, the bacterial count was markedly decreased but in all the slurry added cheeses still had the higher number.
B. Biochemical changes:
B.1. Glycolysis:
- Carbohydrate degradation (Glycolysis) during cheese ripening was determined as the changes in the pH and acidity. As it is believed, the, major part of lactose is degraded during the manufacturing stage and during the two or four weeks during ripening period.
- Lower pH values and higher acidity were observed for the higher fat cheeses.
- During the ripening period, although there is an increase in the acidity and a decrease in pH up to the end of ripening process, the effect of the fat still have the same magnitude between different cheeses.
- A slight decrease in pH and an increase in the acidity were mentioned for the slurry-added cheeses compared to that without added slurry.
- The effect of slurry on the pH and acidity continued up to the end of ripening process.
B.2. Soluble nitrogen content:
- With the decrease in fat content, the fresh cheeses gave greater values for the SN.
- The SN coefficient decreased as the fat content in cheese decreased, cheeses at 1% fat in cheese milk showed 314 to 361 mg N/100 g cheese compared with 259 to 318 mg N/100 g cheeses of 4% fat in cheese milk, these gave SN/TN of 5.45 to 6.56 and 6.46 to 8.01, respectively.
- It can also noticed that addition of cheese slurry increased slightly both SN and SN/TN of cheeses, at all stages of cheese maturation.
- With the progress in ripening period there were a gradual increase in SN and SN/TN in all cheeses, but as it was pridected the cheeses of higher fat content showed greater rate of increase compared with that contain less fat content.
- The quantity of the increase in SN at the end of maturation period were 372, 387, 478 and 475 mg N for A1, A2, A3 and A4 cheeses, respectively.
- Cheeses with added slurry gave higher values for the SN or SN/TN than that without added slurry. At a given fat content, the slurry treated cheese gave higher protein degradation than the untreated cheese, this was noticed extensively as the fat content in cheese increased. The quantity of the increase in SN up to the end of ripening period were: 340, 416, 535 and 508 mg N for S1, S2, S3 and S4 cheeses, respectively. The S3 cheeses revealed rate of proteolysis higher than that of A4 cheeses, which support the effective role of cheese slurry in the improvement of low-fat cheese ripening.
Phosphotungestic acid-soluble nitrogen (PTA-SN):
The results indicated that, at a given fat content, the same effect on the PTA-SN for treated or untreated fresh cheeses was observed.
- Although the cheeses of low-fat contents revealed higher values of PTA-SN than cheeses of higher fat content at all stages of cheese ripening, this behaviour become more pronounced in the case of cheeses made with slurry.
- The cheeses of the lowest fat content treated with slurry (S1) with the highest fat content without slurry (A4), the PTA values at the end of ripening period were: 247 and 164 mg respectively, while the PTA-SN/TN values were: 4.27 and 3.89 respectively.
Polyacrylamide gel electrophoresis (PAGE):
The electrophoretic patterns of Ras cheeses treated with or without slurry at different fat levels appeared to have some protein bands in different regions. The intensity of S1- and -casein bands decreased during ripening. Many bands could be detected in the fast region that could be drived from the degradation of S1-casein, and also, the figure revealed to great degradation of -casein.
The intensity of the bands of low-fat cheeses that was treated with slurry decreased with the progress of ripening period. This result confirmed the level of PTA-SN which was greater at low levels of fat with treated slurry cheeses than the others.
Organoleptic properties:
Flavour, body and texture and appearance of Ras cheese of different fat levels made with or without addition of slurry were examined after 45 and 90 days of ripening. Cheeses with high fat content exhibited higher values for all indices of organoleptic properties at all ripening stages.
Slight effect was found in cheeses with added slurry compared with that without slurry, higher scores were observed for the former along the ripening period.
Although the proteolytic indices (SN, PTA-SN and PAGE) showed differences between cheeses made with added slurry than others, the negligible or undetectable differences in the organoleptic properties between these cheeses might be indicating that panelists had difficulty preceiving these attributes among the cheeses or difficulty interpretation of the terms.