Fermentation of Solid Media from Tempe to Enzymes

Dr. Ir. Budiatman Satiawihardja, M.Sc.

Expert Member of LPPOM MUI,

Lecturer at the Department of Food Technology, Faculty of Agricultural Technology, Bogor Agricultural University

It is known that the fermentation technology consists of fermentation of liquid media and solid media. The following discussion is limited to solid media fermentation in order to make an analogy with the production of tempeh.

During the process of production, Tempe seeds (hereinafter referred to as “inoculum”) in soybeans that have been boiled for a while and have been cooled need to go through the inoculation process. Previously, these soybeans were wrapped in plastic bags or spread on banana leaves and inoculated with a number of mold seeds consisting of a mixture of Rhizopus mold types, including Rhizopus oligosporus and Rhizopus oryzae.

Furthermore, this mixture is incubated at a relatively sterile room temperature which in the room is already dominated by the Tempe-producing mold. About 3-5% of tempeh inoculum, mixed with soybeans evenly.

The amount of mixed inoculum has been reduced a lot (ideally around 10%), because the tempeh fermentation space is usually quite “saturated” with the seeds (inoculum) of the tempeh. In a more traditional way, soybean and inoculum tempe, are contained in banana leaves of the desired size.  

The mixed system of soybean and inoculum is then incubated in a room for about 36-48 hours (in a special place for making tempeh, it is usually 36 hours). After that time, the tempeh can be harvested.

As with making tempeh, enzyme production on solid media can be done in the same way. Some of the common enzymes made using solid media include amylase, pectinase, glucoamylase, cellulase, protease, hemicellulose and lipase. Some solid media commonly used in solid media fermentation include tapioca dregs, bagasse or bagasse, beet pulp, and wheat bran. In general, the process of making enzymes using solid media begins with the preparation of the inoculum. 

The inoculum medium is sterilized first to kill unwanted microbes. To complete the nutrients required for optimum microbial growth, the media is enriched with a source of nitrogen, vitamins and macro and micro minerals. The prepared inoculum is inoculated into the enriched media. Fermentation is carried out for a certain time, generally 36-48 hours. In contrast to tempeh, which is medium and tempeh yeast which are products that can be directly harvested, enzymes need downstream processes in the form of extraction or isolation, purification or purification, and concentration.

Extraction is carried out using water and other solvents. To assist the release of enzymes from solid media, surfactants are used. The extract liquid is separated from the mixture by filtration or centrifugation. Furthermore, the enzyme is isolated or purified by means of a microfiltration membrane. The product obtained is a crude and concentrated enzyme liquid. This product can be used as a commercial enzyme. If the desired form of the final product is in the form of a powder then spray drying can be done.

Depending on the level of enzyme purity to be obtained, the enzyme harvesting stage can be carried out as desired. After this first filtering process it can be continued with ion-exchange filtering.

For the purposes of crude enzyme extract, the first filtering result can be continued by slow evaporation, using low heat until the desired amount of concentrate is obtained. In this crude enzyme extraction process, a mixture of other enzymes is usually obtained in minor amounts.

For example, in producing the pectinase enzyme as the dominant product, enzyme activity testing can be directly carried out. The simplest is to use a pectin solution whose viscosity is measured according to the treatment time by a crude solution of the pectinase enzyme. For the production of other enzymes there are many ways to measure their activity, including the activity of pectinase itself, considering the many types of pectinase enzymes themselves. For example, the polygalacturonate enzyme, the pectin esterase enzyme, the pectin methyl esterase enzyme that may be produced by many types of microbes can degrade pectin into methyl alcohol, polygalacturonate acid, and poly-galacturonic acid and methyl alcohol.

There are two ways to measure enzyme activity. The first is to measure the remaining substrate, and the second is to measure the enzyme product produced. In this case, an adequate knowledge of biochemical component analysis is required. In the first way, for example in the case of starch degradation, we may analyze the remaining starch or its degradation products such as glucose.

The chosen method also depends on a more specific purpose, for example, if you want to what extent the substrate has been degraded, measure the pathway, if you want how far the final product of degradation is, then measure the concentration of glucose produced.

Returning to the case of enzyme production using solid media fermentation techniques, the measurement of the product formed is more common than the measurement of the remaining substrate, because the measurement of the residual substrate does not reflect the desired goal. Therefore, knowledge of measuring small components is more important than knowledge of measuring large components. Fortunately, this is well known. For example, measuring glucose in the case of starch degradation is easier and more specific than measuring the amount of starch that has been degraded or remains. Likewise, measuring the formed galacturonic acid is easier and more specific than measuring the amount of pectin that has been degraded.

Now, what enzymes are widely used and can be produced, such as making tempeh? If I may mention, some examples include amylase (for glucose production), pectinase (for purifying juice), protease (for the production of amino acids), and lipase (for the interesterification process). Indeed, the level of difficulty is different.

The author has tried to sort the examples above according to the level of difficulty to the best of the writer’s knowledge. For those who are interested in biotechnology, let’s try together to develop the above aspects, from upstream to downstream.

In our country, fermentation technology for chemical production at the industrial level, for example, is the production of citric acid in Lampung using solid media fermentation techniques. It is noteworthy that the added mineral solution (as the most likely tipping point aspect) should not contain questionable ingredients such as peptone, protein hydrolyzate or other additives which may raise questions.

For example, peptone, a water-soluble protein hydrolyzate, is formed in the early stages of the protein hydrolysis process. Thus, peptone is still a fairly complex component. The halalness of protein hydrolyzates of course depends on the origin of the protein. If the protein comes from haram animals, of course the law is also haram. If there are additional ingredients other than that, the type must be clearly defined. (***)

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