Investigating fruit juice clarifying enzyme and providing a kinetic model for it

Dissertation for Master's degree ((M.Sc.))

Focus: food industry

Abstract:

Pectinases are a group of hydrolytic enzymes that break down pectic substances. Exopolygalacturonase (exo-p) and endopolygalacturonase (endo-p) are two types of this group. In this study, two fungal species, namely Aspergillus niger and Vetrichodermarisi, which were able to produce pectinase, were isolated. The results of the comparison of the two species showed that the Aspergillus niger fungal species has a higher ability to produce polygalacturonase compared to the Trichoderma species. Optimization of some effective factors in the production of polygalacturonases by Aspergillus niger under discontinuous surface culture conditions showed that the maximum amount of production is obtained at a concentration of 50 g/L of pectin as the only source of carbon and energy. In this case, the optimal ratio of ammonium sulfate to pectin was 0.3, and the optimal fermentation time was 4 days. The results showed that continuous production of polygalacturonase is possible by surface culture fermentation method. When the concentration of pectin in the feed was 50 g/L and the retention time was 1 day, the highest activity of exopolygalacturonase was 1.5 U/mL in the outflow, and under the same conditions, the activity of endopolygalacturonase was about 0.015 U/mL. Using reverse flow with a ratio of 0.8 increased the concentration of exo and endopolygalacturonase up to 1.8 and 0.016, respectively. The production process of polygalacturonases was modeled in the surface culture reactor. For this, a Michaelis-Menton kinetic equation was used. The parameters of the kinetic relationship were obtained using a series of experimental data. The presented model predicts other experimental data with acceptable accuracy.

The replacement of pectin with sugar beet pomace was also investigated in the surface culture method. Galacturonases were cultured in amounts comparable to those produced in Barouche pectin fermentation (5.5 U/g for exo and 0.06 U/g for endopolygalacturonase). Then the production of polygalacturonases from sugar beet pulp as a substrate by Aspergillus niger (A.niger) in solid state fermentation (SSF) was carried out in a semi-continuous manner by using drop by drop mineral solution on the pulp. In this system, the process continued for 25 days and the overall production of pectinase in this system was much higher than the discontinuous system, that is, the cumulative production of the enzyme in these 25 days was U/g24/69 for exopolygalacturonase and U/g738/0 for endopolygalacturonase, which were very favorable results.

Introduction

Currently the world population is more than five billion people. There are different ways to provide the food they need. With the progress of applied microbiology, the use of microorganisms and enzymes derived from them and other microbial metabolites, as methods of food production and improving their quality and quantity, have been introduced in food production. Therefore, by controlling and processing relative methods and using known and suitable microorganisms, the quality and quantity of all kinds of food can be increased significantly. Among them, microbial enzymes such as pectinase, cellulase, amylase and protease can be used to clarify fruit juice and remove pectin and cellulose fibers from it. Pectinase was discovered in 1930 by many experiments that were carried out in the production process of fruit juice and wine. During the 1960s, a lot of information was obtained from the chemistry of plants that showed the effective use of enzymes, one of which was the pectinase enzyme. Fruit juice is the liquid extracted from the cells of ripe fruits. There are three different types of sugar in each type of ripe fruit, namely fructose, glucose and sucrose. Also, in all kinds of fruits, there are various organic acids, including citric acid, malic acid and tartaric acid, along with various vitamins and minerals.These compounds in fruits give them their sweet and sour characteristics. Fruit juices that are of low quality and dark color are offered in the market, although they have a long life and a low price, they are not liked by customers. Therefore, fruit juice producers should pay attention to the quality of fruit juice. One of the important stages in the process of producing fruit juices, especially apple juice, is the enzyme stage.  The enzyme step is of particular importance, because by removing this step in the process, it will produce cloudy fruit juice.

Today, in most parts of the world, fruit juice is prepared and produced in a clarified form. Enzymes, in fact, are biocatalysts of protein nature and are produced by living organisms of animals, plants and microorganisms. All reactions in living cells require enzymes. The main role of enzymes in living organisms is to catalyze decomposition and synthesis reactions. Enzymes increase the speed of biological reactions and are exposed to physical and chemical changes.

The catalytic activity of enzymes is due to their specific protein structure, and their catalytic action is carried out in a specific place of the enzyme called the active site or catalytic site.

As mentioned, enzymes can be prepared from various animal, plant and microbial sources. Commercial enzymes are classified into the following three groups:

1- Industrial enzymes, such as amylases, proteases, glucose isomerase, lipase, catalase, penicillin amylase and pectic enzymes

2- Analytical enzymes, such as glucose oxidase, galactose oxidase, alcohol dehydrogenase, Hexokinase, muramidase and cholesterol oxidase

3- Medical enzymes, such as asparaginase, protease, lipase and streptokinase

Enzymes are divided into 6 categories from the point of view of the types of reactions they catalyze:

The first category, the group of oxidoreductases: these enzymes The catalysts of oxidation-reduction reactions are dehydrogenases, oxidases, and peroxidases. The second category, the group of transferases: these enzymes act as catalysts in reactions in which chemical groups are transferred. In this group, we can mention the kinases that transfer the phosphate group from ATP to another body and the transaminases that are responsible for the transfer of the amino group.

The third group, the group of hydrolases: these enzymes are the catalysts of hydrolysis reactions and include: pectic enzymes, lipases and amylases.

Category Fourth, group of lyases: these enzymes remove specific groups without hydrolysis and form a double bond. From this group, decarboxylases and aldolases can be mentioned. The fifth category, isomerases: these enzymes are the catalysts of the internal displacement reaction on a raw material. From this group, we can mention cis-trans isomers, epimerases and racemases.

In these reactions, ATP will be created as an energy source by water-binding ligases, and AMP and pyrophosphate.

Pectinases are enzymes that are usually produced by microbial methods, as an extracellular product by the microorganisms that produce them. are produced.

Generally, pectinase enzyme is produced by three methods: submerged fermentation [1], solid state fermentation [2] and surface culture [3]. Solid state fermentation is of interest to researchers due to its many advantages compared to the other two methods. Surface cultivation also has advantages that distinguish itself from the other two methods. The most important advantages of this method are: the low cost and energy required, at the end of fermentation, the liquid under the microbial mass can be removed and replaced with a new culture medium, in this way, by preserving the mushrooms from the previous cycle, the average fermentation period in the new cycle is reduced, there is no need for aeration inside the culture medium and stirring. assigned