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Critical Points at Every Stage of the Fermentation Process


1 Budiatman Satiawihardja, 2Sri Mulijani, 3Henny Nuraini, 4Khaswar Syamsu

“In the series of processes to produce a fermented product, there are several stages that generally can cause the products become non-halal

In the series of processes to produce a fermented product, there are several stages that generally can cause the products become non-halal. In other words these stages contain a critical point. The stages are:

– Storage of microbial strains (seeds)

– refreshment step (inoculum)

– Inoculum Development

– Fermentation process

– Product isolation/purification

The following discussion explains the origin of illicit materials that exist at each stage and their critical points if they are associated with the final product.

A.    Storage of microbial strains

Microbial strains, whether self-isolated, or purchased from culture banks are usually stored in freezers form so that they are dormán (in-active) before use. To protect these microbial strains from being damaged during storage at cold temperatures, a protective material (cryoprotectant) is needed. The ingredients commonly used as cryoprotectants are glycerol, lactose, skim milk powder, sterile soil, and others. Glycerol material can be come from the results of hydrolysis of animal fats. Meanwhile, lactose can come from the by-products of cheese processing that can be use animal enzymes. The critical point are source of glycerol and enzymes.

B.     Strain Refreshment Step (slant agar)

Before the microbial strain is used as an inoculum, the microbial strain is first refreshed on the medium to be oblique. Agar tilt, in addition to containing agar from seaweed, is also enriched with nutrients in the form of carbon sources, nitrogen and minerals. Some microbial strains use animal blood as a source of nutrients on oblique agar. The source of carbon used is usually in the form of simple sugars such as glucose / dextrose. The source of nitrogen used is generally in the form of péptides such as peptones which are the result of partial hydrazation of proteins. Proteins can be of animal or vegetable origin, while enzymes used to hydrolyze are generally of animal or microbial origin. Its critical point lies in the source of proteins and enzymes for hydrolyzing.A commonly used enzyme is a protease derived from the pig pancreas (pancreatic enzyme). In media that uses animal blood, the critical point lies in the source of the animal and in the downstream process of the product.

C.    Inoculum Development

Microbes that have been grown on an oblique agar, then inoculated on a liquid medium in an Erlenmeyer (shake flask) that will be used as an inoculum for the next process. The media used in this inoculum is a source of carbon, a source of nitrogen, a growth factor, vitamins and minerals. The carbon source used is generally in the form of sugar (glucose, sucrose, etc.), while the nitrogen umber used is generally in the form of ammonia, ammonium sulfate, urea, etc.In addition, vitamins B1, B6, B complex, and minerals such as KH2PO4, K2HPO4, MgSO4, ZnSO4 etc. are also added. The tipping point lies in vitamins that can be of animal or microbial origin involving animal ingredients. Inoculums are made in a multistage manner on various bioreactor scales to shorten the phase lag on the next bioreactor scale.

D.    Production Media Creation (Commercial Scale)

The production medium is the last medium created in order to produce the desired product. If the production scale is large, then the production medium is made in large quantities, For one production batch, the materials used can be in quantity on a quintal scale or tons in a bioreactor of thousands of liters in size although some kelumit materials can be relatively small (trace minerals, vitamins, hormones). The main media used is usually agricultural by-products that are available in abundant and low-priced quantities, such as molasses as a source of carbon, corn step liquor as a source of nitrogen.

To prevent the formation of abundant foam due to the agitation process of materials containing protein, anti-foaming (antifoam) is usually added. Anti-foaming ingredients are generally surfactants that can come from animal fats or chemicals. Its critical point lies in the source of the antibusa used.

Animal cell culture media usually contains very complex components, contains about fifty single components and is often supplemented with animal blood serum. Serum is a complex component and is expensive. If the product of animal cell culture is a protein, then the purification of the product becomes very complex. Therefore, on a large scale, the composition of the medium is very important to consider so that it is as cheap as possible. So efforts were made to replace serum components with other cheaper ingredients, of course, through various stages of research. These efforts have now paid off, some types of animal cell cultures can now be grown in a medium without containing serum, for example hybridoma cell cultures and CHO (Chinese hamster ovary) cells. The protein concentration in the antibodies produced by hybridoma cells ranges from 100-200 mg / liter, so that the process of purifying from a medium without serum becomes easier.

E.     Product Isolation/Purification

The process of harvesting products from microbial cells is carried out with surfactant auxiliaries such as Tween 80, Span 60. Surfactants can be derived from animal or vegetable fats. The tipping point is in the source of the surfactant used.

At the stage of isolation, the desired product is separated from other unwanted substances. In this process it is likely that the product is precipitated by organic solvents such as ethanol (e.g. in the separation of microbial gum). The tipping point is on the source of ethanol used whether it comes from khmar or from industrial ethanol.

For the decolorization process, activated charcoal is used. Activated charcoal can come from wood, coconut shells, palm shells, bamboo, coal or animal bones. The critical point lies in the source of activated charcoal used.

At the final stage, the product can be formulated using other additives as coating materials. Coating materials can use gelatin, lactose, maltodextrin, dextose mono hydrate, and so on. The critical point lies in the coating agent used.


The fermentation process is a biological process, a life process of living cells that grow or multiply. In this process, there is a conversion or conversion of substances in the medium into metabolite products (internal or external) as well as cell propagation or growth. The resulting metabolites, even if they are not the main product (i.e. by-products), may be substances that can cause intoxication (khamar) so that they can be categorized as critical.

In addition, in the fermentation process there may be the addition of a component during the process (e.g. anti-foam, growth regulator or stimulant, or perhaps other substances that are deemed necessary). Then it is necessary to trace the halalness or monasticity of these substances.

In the fermentation process using plant cells, one of the techniques to obtain products sustainably is to adapt them into the absorbing material, for example resin or activated charcoal; in this case it allows it to be used activated charcoal or gelatin-coated resin.

According to the MUI Fatwa Commission, the use or contact of the material with pig elements and their derivatives at any stage in the fermentation process, will make the product non-halal. But for illicit materials other than pork and their derivatives and unclean, the halalness of the product depends on the downstream process (downstream processing) which can guarantee the separation of the unclean material from the product.


1 Dr. Budiatman Satiawihardja (LPPOM MUI Expert Team)

2 Sri Mulijani (LPPOM MUI Expert Team, Lecturer at the Department of Chemistry IPB)

3 Dr. Henny Nuraini (LPPOM MUI Expert Team, Teaching Staff of the Faculty of Animal Husbandry IPB, Halal Science Center IPB)

4 Prof. Ir. Khaswar Syamsu, MSc. PhD, (Head of Halal Science Center IPB and Coordinator of LPPOM MUI Experts)

Halal Journal 131

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