IMMOBILIZATION AND ESTIMATION OF ACTIVITY OF YEAST CELLS BY ENTRAPMENT TECHNIQUE USING DIFFERENT MATRICES

IMMOBILIZATION AND ESTIMATION OF ACTIVITY OF YEAST CELLS BY ENTRAPMENT TECHNIQUE USING DIFFERENT MATRICES

Nelluri Kanaka Durga Devi ^* and Atluri Satya Sai Nagamani

KVSR Siddhartha College of Pharmaceutical Sciences, Vijayawada - 520010, Andhra Pradesh, India.

ABSTRACT: Background: It becomes clear that living processes were based on the action of enzymes. Cell immobilization serves as a useful tool for the immobilization of intracellular enzymes. The purpose of study is to improve economically important processes by attaching the yeast cells to matrices. Methods: In this analytical study by using mixed models of matrices like (sodium alginate alone, sodium alginate and gelatin, sodium alginate and agar, sodium alginate and glutaraldehyde) to determine the effective combination for immobilizing cells without any damage. Results: It was noted that the glucose content in both cells increased which indicates the production of carbon dioxide and alcohol and then the values started falling down this is because the osmotic concentration of the sugar gets so great that the yeast is unable to get enough water for growth. Conclusion: Glucose utilization was analyzed in free and immobilized cells on four different matrices for verifying their performance and the improvement of immobilized yeast cells over free yeast cells. Use of mixed matrices enhanced the biological activity of cells thus improved their performance. Immobilization of yeast cell showed technical and economic advantages over free cell system.

Free yeast cell, Immobilized yeast cell, Sodium alginate, Gelatin, Glutaraldehyde

INTRODUCTION: Enzymes are macromolecular biological catalysts. Enzymes accelerate, or catalyze, chemical reactions. The molecules at the beginning of the process upon which enzymes may act are called substrates and the enzyme converts these into different molecules, called products ^{1, 2}. Almost all metabolic processes in the cell need enzymes in order to occur at rates fast enough to sustain life. The set of enzymes made in a cell determines which metabolic pathways occur in that cell.

The technique of cell immobilization is an outgrowth of enzyme fermentation, because of their specific catalytic activity and their high performance under mild physiological conditions. Nevertheless, enzyme immobilization process suffers from certain disadvantages which are the cost of pure enzyme, the difficulty of recycling them by extraction and problems of product contamination by leaking from the immobilized enzymes ³. To circumvent these disadvantages, the more readily available microbial enzymes, together with the cells containing them are bound to carriers by various methods, often with remarkable improvements in enzyme activity and half-life.

The present study is not  about carrying out general method of  immobilization this study  deals with various   aspects  such as immobilization of whole cells by entrapment  technique using  different mediums  and estimating their activity differently by  measuring the glucose absorbed by the cells which promote their growth and is responsible for  production  of its  byproducts.

MATERIALS AND METHODS:

Materials: The organism used in present study is Saccharomyces cerevisiae and a commercial grade baker’s yeast was used.  Sodium alginate with other chemicals like gelatin, agar, and glutaraldehyde used were purchased from commercial sources and were of analytical grade. Calcium chloride issued for the formation of beads. The sucrose solution serves as a source of glucose for Saccharomyces cells. The flasks and beakers used in the experiment were maintained in sterile conditions so that, they can maintain consistency in results. For the determination of Glucose presence, the amount of glucose present was measured by glucose strips.

Choice of Support for Immobilisation: The support materials used for immobilization range from elegantly produced spheroids of inorganic oxide to sand and bricks and great ingenuity has gone into devising methods of binding biocatalysts on them ^{4, 5}. Commercial success has been achieved where the support material has been chosen for the flow characteristics. Cost, non-toxicity and immobilization method tailored to give maximum biocatalystic activity while retaining the desirable flow characteristics

Methods:

1. Cell Immobilization by Entrapment in Sodium Alginate: Add 1 gm of sodium alginate to 25 ml distilled water in a beaker. Mix thoroughly. Mix 5 gm of dried yeast in 25 ml distilled water in a beaker and Stir well. Prepare 100 ml of 1.5 gm calcium chloride solution in the large beaker. Add the yeast suspension to sodium alginate solution and mix thoroughly with glass rod. Draw all of the mixture into 20 ml syringe. From a height of 10 cm release the mixture from syringe ⁶, one drop at a time, into calcium chloride solution. Beads containing yeast cells will form. Leave the beads to harden for at least 10 min. Filter the beads through a sieve and rinse with distilled water.

Estimation of Yeast Activity: Mix another 4 gm of yeast in 25 ml distilled water. Pour this yeast into a separating funnel labelled “Free Yeast”. Pour the beads into another separating funnel labelled “Immobilized Yeast. Prepare 100 ml of 1 gm sucrose solution with water warmed to about 40 ºC. Pour 50 ml sucrose solution into the yeast in each of the separating funnel. Using glucose strips   test samples from each funnel for glucose ⁷. Repeat the test at five min intervals. Record result: Run off the remaining product from each funnel into beakers. Compare the turbidity of the solutions.

2. Cell Immobilization by Entrapment in Sodium Alginate and Gelatin: Add 1 gm of sodium alginate to 25 ml distilled water in a beaker and mix thoroughly. Add 1 gm of gelatin to 25 ml distilled water and stir well. Mix 5gm of dried yeast in 25 ml distilled water in a beaker and mix thoroughly. Prepare 100 ml of 1.5 gm calcium chloride solution in the large beaker. Add the gelatin solution to sodium alginate solution and stir well. Add the yeast suspension to sodium alginate ⁸ and gelatin mixture and mix thoroughly with glass rod. Draw all of the mixture into 20 ml syringe. From a height of 10 cm release the mixture from syringe, one drop at a time, into calcium chloride solution. Beads containing yeast cells will for, Leave the beads to harden for at least 10 min and Filter the beads through a sieve and rinse with distilled water.

Estimation of Yeast Activity: Mix another 4 gm of yeast in 25 ml distilled water. Pour this yeast into a separating funnel labeled “Free Yeast”. Pour the beads into another separating funnel labelled “Immobilized Yeast”. Prepare 100 ml of 1gm sucrose solution with water warmed to about 40 ºC. Pour 50 ml sucrose solution into the yeast in each of the separating funnel. Using glucose strips   test samples from each funnel for glucose. Repeat the test at five min intervals. Record result: Run off the remaining product from each funnel into beakers. Compare the turbidity of the solutions.

3. Cell Immobilization by Entrapment in Sodium Alginate and Agar: Add 1 gm of sodium alginate to 25 ml distilled water in a beaker and mix thoroughly. Add 1 gm of agar to 25 ml distilled water and stir well. Mix 5 gm of dried yeast in 25 ml distilled water in a beaker and mix thoroughly. Prepare 100 ml of 1.5 gm Calcium chloride solution in the large beaker ^{9, 10}. Add the agar solution to sodium alginate solution and stir well.

Add the yeast suspension to sodium alginate and agar mixture and mix thoroughly with glass rod. Draw all of the mixture into 20 ml syringe. From a height of 10 cm release the mixture from syringe, one drop at a time, into calcium chloride solution. Beads containing yeast cells will form. Leave the beads to harden for at least 10 min. Filter the beads through a sieve and rinse with distilled water.

Estimation of Yeast Activity: Mix another 4 gm of yeast in 25 ml distilled water. Pour this yeast into a separating funnel labelled “Free Yeast”. Pour the beads into another separating funnel labelled “Immobilized Yeast”. Prepare 100 ml of 1 gm sucrose solution with water warmed to about 40 ºC. Pour 50 ml sucrose solution into the yeast in each of the separating funnel. Using glucose strips   test samples from each funnel for glucose. Repeat the test at 5 min intervals and record the results. Run off the remaining product from each funnel into beakers. Compare the turbidity of the solutions.

4. Cell Immobilization by Entrapment in Sodium Alginate and Glutaraldehyde: Add 1 gm of sodium alginate to 25 ml distilled water in a beaker and mix thoroughly. Add 1 gm of glutaral-dehyde to 25 ml distilled water and stir well.

Mix 5 gm of dried yeast in 25 ml distilled water in a beaker and mix thoroughly. Prepare 100ml of 1.5 gm calcium chloride solution in the large beaker. Add the glutaraldehyde solution to sodium alginate solution and stir well. Add the yeast suspension to sodium alginate and glutaraldehyde mixture ^{11, 12, 13} and mix thoroughly with glass rod. Draw all of the mixture into 20 ml syringe. From a height of 10 cm release the mixture from syringe, one drop at a time, into calcium chloride solution. Beads containing yeast cells will form. Leave the beads to harden for at least 10 min. Filter the beads through a sieve and rinse with distilled water.

Estimation of Yeast Activity: Mix another 4 gm of yeast in 25 ml distilled water. Pour this yeast into a separating funnel labeled “Free Yeast”. Pour the beads into another separating funnel labelled “Immobilized Yeast”. Prepare 100 ml of 1 gm sucrose solution with water warmed to about 40 ºC. Pour 50 ml sucrose solution into the yeast in each of the separating funnel. Using glucose strips test samples from each funnel for glucose. Repeat the test at 5 min intervals and record the results. Run off the remaining product from each funnel into beakers. Compare the turbidity of the solutions.

RESULTS AND DISCUSSION:

TABLE 1: THE AMOUNT OF GLUCOSE PRESENT IN CELLS BY ENTRAPMENT IN SODIUM ALGINATE

TABLE 2:  THE AMOUNT OF   GLUCOSE PRESENT IN CELLS BY ENTRAPMENT IN SODIUM ALGINATE AND GELATIN

TABLE 3: THE AMOUNT OF GLUCOSE PRESENT IN CELLS BY ENTRAPMENT IN SODIUM ALGINATE AND AGAR

TABLE 4: THE AMOUNT OF GLUCOSE PRESENT IN CELLS BY ENTRAPMENT IN SODIUM ALGINATE AND GLUTARALDEHYDE

FIG. 1: PRESENCE OF GLUCOSE BY ENTRAPMENT OF CELLS IN SODIUM ALGINATE       

FIG. 2: PRESENCE OF GLUCOSE BY ENTRAPMENT OF CELLS IN SODIUM ALGINATE AND GELATIN

FIG. 3: PRESENCE OF GLUCOSE BY ENTRAPMENT OF CELLS IN SODIUM ALGINATE AND AGAR         

FIG. 4: PRESENCE OF GLUCOSE BY ENTRAPMENT OF CELLS IN SODIUM ALGINATE AND GLUTARALDEHYDE                                  

DISCUSSION: The range of microbial cells used in immobilization roughly equals that used in fermentation processes. The aim is to improve economically important processes by attaching the microbial cells to matrices.

In the present study attempts were made to carry out whole cell immobilization of Saccharomyces species by gel entrapment in different matrices. The different matrices selected were sodium alginate, sodium alginate and gelatin mixture, sodium alginate and agar mixture, sodium alginate and glutaraldehyde mixture. In Cell immobilization by entrapment in sodium alginate method, the immobilized beads were formed by mixing yeast suspension in sodium alginate solution. The beads obtained by this method are spherical and were stable. From the results in Table 1, it was observed that the glucose content in both immobilized and free yeast reached a threshold value and started falling down. In Cell immobilization by entrapment in sodium alginate and gelatin method, reported that gelatin was used at a concentration of 20% for cell immobilization, hence we employed this concentration of gelatin for entrapment. However it was observed that the bead formation does not takes place.  To promote the bead formation entrapment was done by using sodium alginate and gelatin mixture. Then the beads were obtained.

From the results in Table 2 it was observed that the glucose content in both immobilized and free yeast reached a threshold value and started falling down. In Cell immobilization by entrapment in sodium alginate and agar method, the survey of literature indicated that agar is widely used for immobilizing various microbial cells. Accordingly we attempted to immobilize our cells by gel entrapment using agar. But the beads were not formed. For the formation of beads entrapment of cells were carried in sodium alginate and agar mixture. From the results in Table 3 it was observed that the glucose content in both immobilized and free yeast reached a threshold value and started falling down. In Cell immobilization by entrapment in sodium alginate and glutaraldehyde, the beads were not formed by using glutaraldehyde alone. The use of glutaraldehyde as a matrice or a support for immobilizing enzymes and cells is facilitated by the use of sodium alginate as a cross linking agent. This treatment with sodium alginate resulted in hardening glutaraldehyde leading to improved mechanical strength and stability. From the results in Table 4 it was observed that the glucose content in both immobilized and free yeast reached a threshold value and started falling down.

Yeast is a fungus and needs a supply of energy for its living and growth. Sugar supplies this energy. So we add sucrose solution for the estimation of immobilized and free yeast activity. From all the Fig. 1, 2, 3 and 4, it was noted that the glucose content in both burettes reached a maximum value and started falling down; it may be due to the osmotic concentration of the sugar gets so great that the yeast is unable to get enough water for growth. As fresh yeast is more than 90% water, the single substance most needed for growth is water. Exactly, do not know whether yeast cells are able to take up water actively, by expenditure of metabolic energy to pump the water against the water potential gradient.

When sodium alginate alone was used as matrix the glucose absorbed by beads was little, comparatively the beads in the mixture of sodium alginate and agar has absorbed the maximum amount of glucose than any other mixtures employed in our study. More or less the absorption of glucose by beads entrapped in mixture of sodium alginate and glutaraldehyde were similar to sodium alginate and agar mixture. The beads in the mixture of sodium alginate and gelatin absorbed the glucose at high rate.

CONCLUSION: The use of immobilized whole cells in industrial processes has attracted due to advantages over traditional processes. Immobilization provides high cell concentrations and cell reuse. With the study it is aimed to find the best carrier for immobilization of yeast cells and estimation of their activity with free cells. It was noted that mixed matrices showed better result than the single support so, conducted this analytical study by using mixed models of matrices. Glucose utilization was analyzed in free and immobilized cells on four different matrices.  Use of mixed matrices enhanced the biological activity of   cells thus improved their performance.  Using   free or immobilized yeasts, the rates were of the same order.

ACKNOWLEDGEMENT: The authors are very much thankful to Management of KVSR Siddhartha College of Pharmaceutical     Sciences, Vijayawada for their support and constant encouragement.

CONFLICT OF INTEREST: Nil

REFERENCES:

1. Krajewska B: Chitin and its derivatives as supports for immobilization of enzymes. Acta Biotechnol 1991; 11: 269-77.
2. Jen AC and Wake Mc Mikos AG: Review: hydrogels for cell immobilization. Biotechnol Bioeng 1996; 50: 357-64.
3. Öztop HN, Öztop AY, Isıkver Y and Saraydın D: Immobilization of Saccharomyces cerevisiae on to radiation cross linked HEMA/AAm hydrogels for production ethyl alcohol. Process Biochem 2002; 37: 651-7.
4. Devi KDN and Mrudula SB: Comparison between guar gum and locust bean gum as barrier layers in montelukast press-coated tablets. The Indian Pharmacist 2012; 53-63.
5. Bernt E and Gutmann I: Ethanol determination with alcohol dehydrogenase and NAD. In: Bergmeyer HU, editor. Methods of enzymatic analysis. Florida: Verlag Chemie International 1974; 1499-502.
6. D’Souza SF and Godbole SS: Immobilization of invertase on rice husk using polyethylenimine. J Biochem Biophys Methods 2002; 52(1): 59-62.
7. Gascón S, Neumann NP and Lampen JO: Comparative study of properties of purified internal and external invertase from yeast. J Biol Chem. 1968; 243(7): 1573-77.
8. Strel B, Grba S and Maric V: Enhancement of biomass and fermentation activity of surplus brewers' yeast in a fed-batch process. Applied Microbiology and Biotechnology, 1993; 39: 53-57.
9. Smidsrød O and Skjåk-Bræk G: Alginate as immobilization matrix for cells, Tibtech 1990; 8: 71-78.
10. Devi NKD, Sindhura A and Mrudula BS: The state-of-the-Art Microencapsulation technology: An Overview. Pharma Bio World 2011; 60-65.
11. Wen-tao Q, Wei-ting Y, Yu-bing X and Xiaojun M: Optimization of Saccharomyces cerevisiae culture in alginate–chitosan–alginate microcapsule, Bio-chem. Eng. J 2005; 25, 151-157.
12. Stringer JL and Peppas NA: Diffusion of small molecular weight drugs in radiation-cross linked poly (ethylene oxide) hydrogels. J. Control Re-lease. 1996; 42: 195-202.
13. Nagashima, M, Azuma M and Noguchi S: Technology developments in biomass alcohol production in Japan: continuous alcohol production with immobilized microbial cells, Ann. N.Y. Acad. Sci., 1983; 413: 457.
    

    ---

    How to cite this article:

    Devi NKD and Nagamani ASS: Immobilization and estimation of activity of yeast cells by entrapment technique using different matrices. Int J Pharm Sci Res 2018; 9(7): 3094-99. doi: 10.13040/IJPSR.0975-8232.9(7).3094-99.

    ---

    All © 2013 are reserved by International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.

    ---