الفهرس 
REVIEW OF LITERATUREOnly 14 pages are availabe for public view
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Abstract
The purpose of this study is to genetically improve folate productivity from selected two LAB, namely Lb. acidophilus and B. longum, through UV irradiation, in addition to perform a molecular comparison of mutants between them and wild strains using sodium dodecyl sulfate polyacrylamide gel electrophoresis of total cellular proteins (SDS- PAGE). Using RT- PCR amplification of this FolE gene, one of the key genes in the examined bacterial cells’ folate synthesis pathway, folate productivity and anti-inflammatory activity in both wild strains and four mutants were previously genetically confirmed.
The best mutants were chosen based on their folate productivity, ability to produce starter using both the mutant and wild strains, and ability to manufacture functional fermented milk that was kept at 4ºC for ten days. Following three, seven, and ten days of cold storage, samples were obtained and subjected to chemical, microbiological, and rheological analyses.
The results can be summarized as follows:
Part I: Improving the folate productivity and anti-inflammatory activity of some lactic acid bacteria strains of using UV irradiation mutagenesis: -
• A survey was conducted of the strains (10 Bifidobacterium strains and 17 Lactobacillus strains) expected to produce folic acid and select of the folic acid production strains and verifying the effectiveness of the production of folic acid by some selective strains.
• In the present experiments, the ability of (27 strains) were tested to produce vitamin B9 (folate). The results presented showed that all the tested strains were not folate producers except that Lb. acidophilus ATCC 4356, B. longum ATCC 15708, B. pseudocatenulatum ATCC 27919, Lb. reuteri ATCC 23272 and Lb. plantarum ATCC 8014.
• Productivity was improved genetically for the two strains through UV mutagenesis for various time periods of 5, 10, 15, 20 and 30 sec, and that surviving ratio in cells was inversely correlated with the length of UV exposure.
• The result indicated that there were significant differences between Lb. acidophilus (2) and B. longum (7) counting with increase of UV exposure time.
• The total bacterial count (%) of Lb. acidophilus (2) strain after UV treatment par 5 sec, 10 sec,15 sec, 20 sec and 30 sec in comparison to wild (before UV treatment) is 70.99, 60.73, 60.45, 51.07 and 39.45 % respectively. Also, B. longum (7) decreased in comparison to wild is 61.48, 48.83, 34.45, 33.22 and 28.37 % respectively.
• Both strains showed a gradual increase in the folic acid production with increasing the growth medium initial pH till a certain level then the activity decreases with increasing the pH value, at pH values 4, 5, 6, 7, and 8, Lb. acidophilus had folic acid levels (µg/ml) of 93, 190, 298, 353 and 212, respectively. Additionally, the B. longum levels were 50, 161, 331, 468, and 290.
• The outcome showed that the folic acid concentrations of B. longum (7) and Lb. acidophilus (2) varied significantly depending on the pH level (p ≤ 0.05).
• The two strains responded differently towards the growth temperature. Both 25 and 45°C incubation temperatures caused suppression of folic acid production by Lb. acidophilus and B. longum, and about half of the activity was expressed when incubated at 30° C, while the maximum production was recorded when grown at 37 °C.
• The levels of Folic acid for Lb. acidophilus at incubation temperatures 25, 30, 37 and 45°C were 91, 247, 351 and 280 µg/ml respectively. Also, B. longum levels were 81, 270, 460 and 190 µg/ml respectively.
• The result indicated that there were significant differences between Folic acid concentration of Lb. acidophilus (2) and B. longum (7) at different incubation temperatures.
• There was detectable effect of the inoculum size on the folic acid production of Lb. acidophilus and B. longum, when the cell free supernatants inoculated with different inoculum sizes 50, 100, 150 and 200 μl / 10 ml MRS broth were tested. While giving its maximum folic acid productivity with inoculum size 200 µl.
• Folic acid levels for Lb. acidophilus were 121, 250, 270 and 345 µg/ml at inoculum sizes 50, 100, 150 and 200 µl. Additionally, the B. longum levels were in that order, 98, 240, 300, and 470 µg/ml.
• The result indicated that there were significant differences between Folic acid concentration of Lb. acidophilus (2) and B. longum (7) at different inoculum size (µl) of growth medium.
• Twenty-five mutants from each wild strain tested as improved grown positive mutants. Beside 5% CO2, other optimal conditions for folate production by the mutants were applied, pH 7, temp 37OC and inoculum size 200µl.
• The best mutants in their folate productivity from B. longum were G5 with 945 µg/ml (198%), followed by K1 with 649 µg/ml (136.3%) of wild strain, which was 476.1 µg/ml.
• In case of Lb. acidophilus which gave folate productivity about 365 µg/ml (the wild), the best producer mutants were C4 and A2 with folate ratio of concentrations 1056 µg/ml (289.3%) and 749 µg/ml 205%, respectively.
• The protein level of COX-1 remarkably decreased in A2 mutant with value of 8.4 against to 9.3 μg/ml in wild strain and 13.5 μg/ml in case of Celecoxib. In a different mutant of the same original strain, 8.6 g/ml, it similarly fell but remained marginally higher than those of A2 and C4. While the COX-1 values of the other two B. longum mutants, G5 and K1, were greater than those of A2 and C4, they were still lower than those of Celecoxib and their wild. They were respectively 9 and 9.2 μg/ml.
• The protein level of COX-2 was decreased less than its value with Celecoxib near to be two times of case of both wild strains (B. longum and Lb. acidophilus) with values of 0.041 and 0.037μg/ml respectively. Protein values of COX-2 were decreased more when 4 mutants applied to it, where it reached 0.039, 0.037, 0.035, and 0.030 μg/ml with G5, K1, A2 and C4 respectively.
• Four mutants displayed FolE gene expression folds more than both of their two wild strains when the expression of the FolE gene was measured using folds obtained from the 2^ ddct method; the highest mutant was A2 with 1.46 folds, followed by K1 with 10.3, G5 with 0.97, and C4 with 0.68 folds.
• SDS-PAGE analysis of four mutants and their two wild strains showed protein bands that range from 5 to 245KDa. Mutant G5 that represents in band 3 and mutant K1 that represents in band 6 showed total protein bands 15 and 14 respectively which differ from their wild that in band 5 with 13 bands. G2 has a single band with size 23KDa. In case of L. acidophilus strain which numbered 5 in the order and its two mutants A2 and C4 in order 1 and 2 in gel; mutants A2 and C4 have total protein bands 13 and 15 respectively, while parental strain was 13, A2 and C4 have the single band at 23KDa, whilst the wild didn’t. A2 also doesn’t include bands at 215 and 84KDa while both C4 and wild have at these molecular weights.
• Folic acid’s binding mechanism demonstrated an energy binding of -8.65 kcal/mol against DHFR. In Ala9, Ile7, Phe34, and Ile60 produced four Pi-alkyl, Pi-sigma, and Pi-Pi interactions with folic acid. Moreover, folic acid connected with Glu30 and Asn64 by three hydrogen bonds that had 1.77, 1.76, and 1.96.
Part II: Production and properties of functional fermented milks from the wild strains and their mutants:
• The results illustrate that culture growth and acidity development (%acidity) is monitored during incubation till coagulation of milk with B. longum, Lb. acidophilus strains and their mutants when grown in skim milk at 37°C. Also, the coagulation time of Lb. acidophilus, B. longum and their mutants are monitored.
• All six strains of lactic acid bacteria grew well at 37°C in 10% reconstituted skim dry milk, also showed that these strains reached the stationary phase of both wild strains (B. longum and Lb. acidophilus) with 34, 40 hr. respectively. While decreased when four mutants applied to it, where it reached 16, 15, 14, and 26 hr. with G5, K1, A2 and C4 respectively.
• The count of all strains (B. longum, Lb. acidophilus) reached 109 CFU /ml at the end of incubation.
• The acidity (%) values ranged from 0.7 to 0.75 in all strains. There is no difference between the wild type strains (Lb. acidophilus, B. longum) and their mutants’ strains in acidity values.
• The coagulation time during incubation in skim milk at 37°C with Lb. acidophilus (2), C4, A2, B. longum (7), K1 and G5 were 40, 26, 14, 34, 15, 16 hr respectively. As shown decrease in coagulation time during incubation in skim milk media at 37°C for C4 and A2 in comparison to wild strain Lb. acidophilus is 65 and 35 % respectively. Also, K1 and G5 decreased in comparison to wild strain B. longum is 44.1 and 47.1 % respectively.
• The data indicated that, total solids contents of different fresh functional fermented milk were 14.06, 13.32, 13.26, 13.96, 13.29 and 13.23% in product made using Lb. acidophilus (2), C4, A2, B. longum (7), K1 and G5 strains respectively.
• The results indicated that type of starter culture used in the production with different treatments and modified strains with UV irradiation mutagenesis did not effect on the total solids content in the different functional fermented milk products.
• The fat contents of different fresh functional fermented milk were made using wild and their mutant strains were 3.2, 3.2, 3.2, 3.3, 3.2 and 3.1% for Lb. acidophilus (2), C4, A2, B. longum (7), K1 and G5 respectively. Generally, the ash and protein content varied from 0.81 to 0.84%, 3.51 to 3.6% and 3.9 to 4.2 % respectively in fresh functional fermented milk made using wild and their mutant strains.
• The data showed that there were non-significant differences of total solids, protein, fat, ash content between fresh functional fermented milk made using the wild strains (Lb. acidophilus and B. longum). Also, there are no significant differences of total solids, protein, fat, ash content between fresh functional fermented milk made using mutant strains (C4, A2, K1 and G5).
• The data indicated that, total lactose contents of different fresh functional fermented milk were 4.2, 3.81, 3.82, 4.1, 3.8 and 3.78% in products made using Lb. acidophilus (2), C4, A2, B. longum (7), K1 and G5 strains respectively. Also, the data showed that there were significant differences of lactose content between fresh functional fermented milk made using the wild strains (Lb. acidophilus and B. longum) compare with fresh functional fermented milk made using mutant strains (C4, A2, K1 and G5).
• According to the data, the acidity and pH values of fresh functional fermented milk produced with the wild strains (Lb. acidophilus and B. longum) did not differ significantly from one another. Furthermore, fresh functional fermented milk produced with mutant strains does not significantly differ in pH. While there were non-significant differences of acidity and pH values between all fresh functional fermented milk produced with mutant strains.
• Although, the data showed that there were significant differences of acidity and pH values between fresh functional fermented milk made using the wild strains (Lb. acidophilus and B. longum) compare with fresh functional fermented milk made using mutant strains (C4, A2, K1 and G5) when fresh and during storage period.
• Generally, the pH values decreased, and the TA gradually increased in all treatments and wild samples during the storage period, this may be due to the activity of fermented milk cultures and because of the activity of fermented milk culture.
• Type of Starter culture significantly affected the SN/TN content in all functional fermented milk samples. The SN/TN content (%) increased slightly in all functional fermented milk treatments as the cold storage increased to 10 days.
• The data showed that significant differences (p < 0.05) were recorded in SN/TN content between samples made using mutant strains and samples made of wild strains when fresh and during storage. The SN/TN content was significantly higher in the functional fermented milk made using mutant strains than in all other treatments. Although the storage period did not significantly affect the SN/TN content in all functional fermented milk samples.
• The data showed that folate levels decreased at a rate of 9.7, 7.9, 9.9, 8.1, 7.4, 10.2% for the first week and decreased gradually throughout the 10-day storage period.
• The result indicated that there were significant differences in folic acid content between all fresh functional fermented milk produced with the wild strains (Lb. acidophilus and B. longum) and their mutant strains. While the storage period of different products made using by wild strains (Lb. acidophilus and B. longum) and their mutant strains (A2, K1, C4, G5) at 4°C had significant effect on folic acid content after 10 days compared to the folate content of fresh functional fermented milk.
• Also, the data showed that the UV irradiation mutagenesis technique improves folic acid production.
• Acetaldehyde content of fresh functional fermented milk samples varied from 212.13 to 295.12 µml/100 g followed by gradual decrease to range from 113.90 to 181.30 µml/100 g at the end of storage period, while diacetyl content ranged from 10.66 to 16.71 µml/100 g in fresh functional fermented milk and decreased to a range from 8.7 to 13.95 µml/100 g at the end of storage period.
• Generally, acetaldehyde content gradually decreased in all functional fermented milk samples as the storage period progressed. While diacetyl content followed by increase in first weeks and gradual decrease until the end of the storage period (10 days).
• There were significant differences in diacetyl and acetaldehyde contents between all treatments. Moreover, acetaldehyde contents were significantly higher in A2, G5, C4 and K1 functional fermented milk samples and diacetyl contents were significantly higher in A2, G5, C4 and K1 functional fermented milk samples. When samples prepared using mutant strains were compared to samples made using wild strains, both fresh and during storage, significant variations (p < 0.05) were noted in the levels of acetaldehyde and diacetyl. Compared to all other treatments, the functional fermented milk produced with mutant strains had noticeably greater levels of acetaldehyde and diacetyl. Also, the storage period had a significant impact on acetaldehyde and di-acetyl contents compared with the fresh samples.
• The Lb. acidophilus count in fresh functional fermented milk made by wild strain (Lb. acidophilus (2)) and their mutant (A2 and C4) strains were 9.06, 8.78 and 9.05 log cfu/ ml respectively, while at the end of storage period, lactic acid bacterial counts decreased to 8.15, 7.84 and 8.01 log cfu/ ml respectively.
• Also, the B. longum count in fresh functional fermented milk made by wild strain (B. longum (7)) and their mutant (K1 and G5) strains were 8.69, 8.4 and 8.49 log cfu/ ml respectively, while at the end of storage period, lactic acid bacterial counts decreased to 7.71, 7.47 and 7.59 log cfu/ ml respectively.
• The gradual decrease in probiotic strains counts was due to the sensitiveness of this bacteria to acid development along the storage period.
• In general, the data showed that there were non-significant differences of Lb. acidophilus and B. longum count between fresh functional fermented milk made using the wild strains (Lb. acidophilus and B. longum) and fresh functional fermented milk made using mutant strains. While the count of Lb. acidophilus and b. longum count decreased significantly at the end of all functional fermented milk storage period compared to the fresh functional fermented milk.
• The results indicated that the counts of LAB in all functional fermented milk maintained acceptable levels (107 -108 CFU / ml) to be considered as functional foods until the end of the cold storage.
• Yeast and mold counts could not be detected in all fresh functional fermented milk samples. Moreover, yeast and mold counts could be observed and counted after 7 days of storage in all functional fermented milk samples.
• Furthermore, coliform bacteria couldn’t be detected in all treatment samples when fresh and during the storage period.
• As seen, all flow curves showed a non-linear course and didn’t start from the origin point (zero shear stress), which indicates a non-Newtonian behavior of pseudo-plastic type.
• Taking rheological parameters of power low model in consideration, sample 2 (wild sample) showed the highest K-value (0.422) compared with the K-value of the samples prepared by using its mutant strains (A2 and C4), where they reported K-value of 0.1478 and 0.222 Pa.sn.
• On other side, K1 and wild sample 7 samples showed K-values of 0.1996 and 0.0856 Pa.sn respectively, being lower than the K-value of G5 (0.2066 Pa.sn).
• All values of flow behavior index (N-values) were lower than unity, being in the range of 0.4603-0.623.
• All functional fermented milk samples showed an initial shear stress in the range of 0.8106-2.468 Pa, which indicates the relativity gel character of the prepared fermented milk samples. Analysis of the rheological data according to the Newtonian model gave lower R2 values being in the range of 0.95-0.984, which prove that the milk sample did not obey the Newtonian model. However, the calculated dynamic viscosity gave values close to those of the apparent viscosity.
Thus, it could be concluded and recommend that modern biological technologies (UV Irradiation Mutagenesis) can be used to develop probiotic dairy products by improving their productive qualities and anti-inflammatory activity, as was done in our study by improving the production of the vitamin folic acid. The use of mutants resulting from mutagenesis by ultraviolet irradiation has also proven to be highly effective as microbial starters to produce functional fermented milk products (chemically, microbially, and rheologically).