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1、The study on the relationship of invertase activity and leavening ability in sweet dough Cui-ying Zhang, Dong-guang Xiao*, Man Xu Key Laboratory of Industrial Microbiology, Ministry of Education, Tianjin Industrial Microbiology Key Laboratory, College of Biotechnology Tianjin University of Science a
2、nd Technology 1038 Dagunan Road, Tianjin 300222, P. R. China *Corresponding author: AbstractThe leavening abilities in sweet dough and the invertase activities were determined in several strains of Saccharomyces cerevisiae to investigate their correlations. There was a general tendency that the hig
3、her invertase activity yeast cells showed, the less leavening ability the strain possessed in sweet dough containing sucrose. The changes of glucose contents detected in high sucrose model liquid dough (HSMLD) mediums further confirmed that strains with higher invertase levels were exposed to high o
4、smotic stress faster and greater in HSMLD mediums than strains with less invertase activities, thus causing that their CO2-producing capacities were inhibited seriously. Then a mutant BM-8 was obtained from bakers yeast strain BY-6 mutated by ultraviolet irradiation. Its invertase level was reduced
5、by 24.9%, while the fermentative activity was proved to be increased by 22.9%. The changes of glucose contents of the mutant BM-8 and its parent BY-6 in HSMLD mediums indicated that compared to the parent BY-6, the mutant BM-8 exposed itself to a less osmotic stress condition during the whole fermen
6、tation process. These results showed that the less invertase levels is undoubtedly helpful for the improvement of leavening ability of bakers yeast and BM-8 was an excellent strain useful in bread production. Keywords-bakers yeast (Saccharomyces cerevisiae); invertase; leavening ability; sweet dough
7、; mutant; glucose content I. INTRODUCTION As the changes of tastes among general population, bakery products such as bread, cake are usually sweetened, sometimes even up to 30% of added sucrose per weight of flour is contained in sweet bread dough, which undoubtedly makes the yeast fermentation acti
8、vity be inhibited by severe osmotic stresses exerted 1-3. In practice this means that yeast inocula have to be at least doubled to achieve a workable rate of leavening when manufacturing sweet baked goods 4. In order to improve the fermentative activities of yeast strains in high sucrose concentrati
9、on media, it is necessary to study on the high sucrose tolerance of bakers yeast. Besides two important osmoprotectants, the content of glycerol 35-7 and trehalose level 67 high sucrose tolerance of yeast strain was also involved in invertase activity 89. It is generally accepted that the extracellu
10、lar invertase (-D-fructosidase) excreted by Saccharomyces cerevisiae cells hydrolyzes sucrose into glucose and fructose, which doubles the osmotic pressure around the cells. Invertase is encoded by one or several SUC genes (SUC1 to SUC5 and SUC7). It has been found that SUC2 is the most common loci
11、in almost all S. cerevisiae strains 1011. These SUC genes generate two different mRNAs: a larger transcript encoding an extracellular invertase with a signal sequence, and a shorter transcript encoding an intracellular form of the enzyme becauce of lack of the signal sequence 12. So far, though sign
12、ificant improvements have been gained in understanding the molecular mechanisms involved in the repression of SUC expression, it is not viable for breeding mutant with less invertase activity at the molecular level. In this work, we investigated the effects of invertase activities on fermentative ab
13、ilities of several S. cerevisiae strains. Also a mutant BM-8 was obtained from bakers yeast strain BY-6 mutated by ultraviolet irradiation. II. MATERIALS AND METHODS A. Yeast strains and culture conditions S. cerevisiae bakers strains, referred to as BY-6, BY-12, BY-14, BY-15, BY-16, A-6, respective
14、ly, used in this study were obtained from the Yeast Collection Center of Tianjin Key Laboratory of Industrial Microbiology. Another seven strains, BM-8, BM-20, BM-26, BM-36, BM-48, BM-60, BM-66, were mutant derivatives of the strain BY-6 created by UV treatment. The yeast strains were routinely cult
15、ured on solid and in liquid YEPD medium containing 10 g/L yeast extract, 20 g/L bacteriological peptone, and 10 g/L glucose. The culture temperature was 30C. High sucrose resistance was assessed using HSuc-solution, which contains 5 g/L yeast extract, and 500 g/L sucrose. Glucose content was measure
16、d in high sucrose model liquid dough (HSMLD) medium, which was modified according to Hino et al. 13 and Jiang et al. 14, contains in g/L: sucrose 300, (NH4)2SO4 2.5, urea 5, KH2PO4 16, Na2HPO4 5, MgSO4 0.6, nicotinic acid 0.0225, Ca-pantothenate 0.005, thiamine 0.0025, pyridoxine 0.00125, riboflavin
17、 0.001, folic acid 0.0005. B. Leavening ability in sweet dough The sweet dough leavening capacity of the yeast was determined following the method described previously by 978-1-4244-4713-8/10/$25.00 2010 IEEEBY-6A-6BY-16BY-12BY-15BY-140510152025303540455005101520253035Leavening ability (mL/2 h)Inver
18、tase activity(U/g dry weight)Rincn et al. 15 and Rollini et al. 16. The reported data represent the mean values of two replicate samples. C. Invertase assay The invertase assay was performed by 3,5-dinitrosalicyIic acid (DNS) method to test the reducing sugar. The reaction mixture for determining ex
19、tra-cellular invertase contained 200 mM sodium acetate-acetic acid buffer (pH 5.2), 100 mM sucrose, distilled water and the appropriate amount of intact cells in a total volume of 0.5 mL. After incubation at 30oC for 5 min, the reaction was stopped by the addition of 3,5-dinitrosalicylic acid reagen
20、t, and the reducing sugars produced in the reaction mixture were measured by the absorbance at 540 nm. One unit of the invertase activity represent 1 mg of glucose released from sucrose with 1 g of yeast (dry weight) for 1 min. D. Determination of high sucrose resistance To determine the high sucros
21、e resistance, 1 mL of yeast cells grown to log phase (approximately OD600=1.0) in YEPD medium were inoculated into a tube containing 10 mL of HSuc-solution and an inverted Durham fermentation tube, where the gases generated during yeast fermentation in Durham fermentation tube were detected after in
22、cubation for 12 h, 16 h, and 20 h at 30oC. E. Measurement of glucose content Yeast cells grown to log phase (approximately OD600=1.0) at 30oC in YEPD medium were harvested by centrifugation at 4,000 g for 8 min. 2 g (wet weight) of cell pellets were inoculated into HSMLD medium. Then glucose in HSML
23、D medium was measured with a Biosensor Analyzer (model SBA-40C, supplied by Biology Research Institute of Shandong Academy of Sciences, China). F. Cell viability assay in hyperosmotic pressure Yeast cells grown to log phase (approximately OD600=1.0) at 30oC in YEPD medium were harvested by centrifug
24、ation at 4,000 g for 8 min, washed with ice-cold water and resuspended in HSuc-solution at a concentration of 1 107 cells/ml, then incubated at 30oC for 3 h with shaking at 150 rpm. Non-treated cells severed as a control. Treated and non-treated cells were diluted 103 and 104 times, respectively. 0.
25、2 ml of the cell suspension was immediately plated on YEPD agars. After incubation for 2 days at 30oC, the colony-forming units (CFU) were counted and the percentage of the CFU of the treated suspension divided by the CFU of the untreated suspension (the total CFU) was defined as the percentage of s
26、urvival. G. Isolation of mutant Mutants with less invertase activities were screened from large numbers of mutagenized cells according to the procedure using glucose oxidase and horseradish peroxidase to assay colonies in situ described previously 17 with small modifications. Yeast cells grown to lo
27、g phase (approximately OD600=1.0) at 30oC in YEPD medium were harvested by centrifugation at 4,000 g for 8 min, washed twice with sterilized saline water, and resuspended in sterilized saline water at a concentration of 1 106 cells/ml. Then 4 mL of the cell suspension was placed into sterilized Petr
28、i dishes (9 cm in diameter) and exposed to UV-irradiation at 254 nm at a distance of 30 cm for 60 s. Typically, the survival of the mutagenized cells was 20%. 0.2 mL of the cell suspensions was plated out on YEPD medium without glucose and incubated in the dark at 30oC to allow them to grow in a col
29、onial mode. After 1 to 2 days of growth the colonies were ready for screening. We added 10 ml of a screening medium, which contained 0.2 g of sucrose, 0.1 g of o-tolidine, 0.2 g of agar powder, 0.3 ml glucose oxidase (100 U) and 0.5 ml horseradish peroxidase (2.5 U) in 100mL sodium acetate buffer (2
30、00 mM), pH 5.2. The colony containing invertase activity developed a dark blue halo of oxidized o-tolidine within 45 min. Mutants lacking invertase remained without a halo. Colonies showing no halo were picked and retested using the screening procedure to ensure that they were invertaseless. Followi
31、ng the secondary screening the mutants were further characterized individually. III. RESULTS AND DISCUSSION A. Invertase activity was inversely related to leavening ability in sweet dough The leavening abilities in sweet doughs, the invertase levels and the high sucrose resistances in six strains of
32、 bakers yeast were measured to analyze their relationship. According to the data shown in Fig. 1, there was a general tendency that the yeast cells showing higher invertase activity usually possess less leavening ability in sweet dough containing sucrose, which was consistent with the fact that the
33、ability of S. cerevisiae to ferment high sucrose concentrations (including sweet bread doughs) is inversely related to the activity of invertase 91819. While, as shown in Table I, among the six strains of bakers yeast, the high sucrose resistance of BY-14 was the strongest, while the leavening abili
34、ty in common sweet dough of BY-14 was the lowest. The results indicated that the leavening abilities in common sweet doughs of different yeast strains were not related to their high sucrose resistances. Figure 1. Leavening ability in sweet dough and invertase activity of different bakers yeast. The
35、leavening abilities of the yeasts were determined by the increases of the volumes of the sweet doughs for 2 h at 30oC. The data represent the mean values of two replicate samples. The invertase was assayed by measuring the glucose released by the action of invertase on sucrose. One unit of the inver
36、tase activity represent 1 mg of glucose released from sucrose with 1 g of yeast (dry weight) for 1 min. TABLE I. HIGH SUCROSE RESISTANCE OF DIFFERENT BAKERS YEASTA Strain Incubation time (h) 12 16 20 BY-6 1 + + + 2 + + + 3 + + + 4 + + + 5 + + +b BY-12 1 + + + 2 - - - 3 - - + 4 + + + 5 - - + BY-14 1
37、+ + + 2 + + + 3 + + + 4 + + + 5 + + + BY-15 1 - + + 2 - - + 3 - - + 4 - + + 5 - - + BY-16 1 - - - 2 - - - 3 - - - 4 - - - 5 - - - A-6 1 + + + 2 + + + 3 + + + 4 + + + 5 + + + aData represent gases generated during yeast fermentation in Durham fermentation tube. The datas of five replicate samples wer
38、e shown. b+ represent that gases were full in Durham fermentation tube. B. Comparison of changes of glucose contents between BY-6 and BY-14 in HSMLD medium To further investigated the effect of the invertase on the fermentative activity of yeast in sweet dough, the changes of glucose contents in two
39、 strains, BY-6 and BY-14, which had the wildest gap in the invertase activities were detected in HSMLD medium. As shown in Fig. 2, the glucose contents in both strains climbed dramatically before 200 mins and then declined slightly in the remaining time. In addition, more glucose was accumulated in
40、BY-14 with the largest invertase level of 46.71 U (Fig. 1), and the maximum of glucose content (12%, wt/vol) was also found to be reached faster than that in BY-6, while the leavening ability in BY-14 (5.5 mL/2h) was significantly less than that in BY-6 (30.5 mL/2h) (Fig. 1). The results indicated t
41、hat in sweet dough containing high concentrations of sucrose, bakers yeast strain with higher invertase level could expose itself faster and to greater osmotic stress than the strain with less invertase activity, thus causing their CO2-producing capacity be inhibited seriously. Therefore, breeding o
42、f bakers yeast strain with less invertase level could be helpful for enhancing the fermentation ability of high sugar dough. Figure 2. Glucose contents in BY-6 and BY-14 during fermentation in HSMLD medium. Yeast cells grown to log phase (approximately OD600=1.0) at 30oC in YEPD medium were harveste
43、d and inoculated into HSMLD medium. The glucose content was measured with a Biosensor Analyzer. TABLE II. INVERTASE ACTIVITIES OF MUTANTS AND THE PARENT BY-6 Strain BY-6 BM-8 BM-60 BM-20 BM-36 BM-26 BM-48 BM-66Invertase activity (U/dry weight) 15.53 11.6712.20 12.99 13.00 13.29 13.49 13.57 Relative
44、invertase activity (%) 100 75.14 78.56 83.64 83.71 85.58 86.86 87.38 TABLE III. THE COMPARISON OF FERMENTATION PERFORMANCE IN DIFFERENT YEAST STRAINS Strain BY-6 BM-8 BM-26 BM-48Leavening ability in sweet dough (mL/2 h) 35 43 38.5 35.4 Average growth rate (h-1) 0.3152 0.37320.3200 0.3520Viability in
45、 hyperosmotic pressure (%) 61.2 75 72.5 70 C. Screening of mutants The bakers yeast strain, BY-6, possessed the lowest invertase activity, and was used as the parent for breeding. After UV mutation, 63 mutants were originally selected by adopting the color developing plates including double mediums,
46、 composed of YEPD medium without glucose and o-tolidine screening medium. In yeast cells, glucose oxidase catalyzes the oxidation of -glucose to produce gluconic acid and H2O2, H2O2 catalyzed by catalase oxidizes o-tolidine, and then o-tolidine changes from colorless to colored. Generally speaking,
47、the contents of colored complex is proportional to the quantity of glucose. So the color developing plate could be used as the original screening method for mutants with less invertase activities 20. Then a secondary screening was performed by directly determining invertase activities. Mutants with
48、invertase activities reduced by more than 10% compared to the parent BY-6 were shown in the Table II. Among 7 mutants, the invertase activity of BM-8 was the lowest. D. Fermentation performance in mutants The leavening ability in sweet doughs, the specific growth rates and the viability in hyperosmo
49、tic pressure of 7 mutants were also detected to screen a superior yeast strain with lower invertase level. The results indicated that BM-8 was an excellent strain. As shown in Table II, the invertase activity of BM-8 was 11.67 U. Compared with the parent BY-6, the invertase activity of BM-8 reduced
50、by 24.9%. The leavening ability of BM-8 in sweet dough was 43 mL/2 h, which increased by 22.9% compared to that of the parent BY-6 (Table III). E. Analysis of change of glucose contents in BM-8 To further verify the effect of invertase activity on the leavening ability in sweet dough, the changes of
51、 glucose contents of both strains BY-6 and BM-8 incubated in HSMLD medium were detected. As shown in Fig. 3, little glucose was accumulated with a slower rate in BM-8 than in the parent strain BY-6. Thus, BM-8 exposed itself to a less osmotic stress condition during the whole fermentation process. T
52、he results demonstrated that less invertase levels in bakers yeast strain was helpful for alleviating the deteriorating trend around the cells, and accordingly improving leavening ability in sweet dough. IV. CONCLUSION In this work, leavening abilities in sweet doughs and invertase activities were d
53、etermined in several bakers yeast strains. The resluts confirmed the effect of invertase activity of bakers yeast on the leavening ability in sweet dough. Because of the hydrolysis of sucrose into glucose and fructose, osmotic pressure around yeast cells doubled. Bakers yeast strain with higher inve
54、rtase level was exposed itself faster and to greater osmotic stress than the strain with lower invertase activity in high sucrose model dough, thus its leavening ability in sweet dough would be further inhibited in a more rapid rate. The invertase level of bakers yeast is reduced to a degree, which
55、is undoubtedly helpful for the improvement of leavening ability of the strain. Thus a mutant BM-8 was obtained, its invertase level was reduced 24.9%, the fermentative activity was also proved increased 22.9%. The results indicated that BM-8 was an excellent strain useful in bread production. ACKNOW
56、LEDGMENT This work was supported by the research fund for Doctoral Programs of Higher Education, China (No. 20050057001) and National Key Project of Scientific and Technical, China (No. 2007BAK36B04). Figure 3. Glucose contents in BY-6 and BM-8 during fermentation in HSMLD medium. Yeast cells grown
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