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Points to ponder while scaling up in Probiotics production

Updated: Dec 14, 2021


Probiotics are live microbial fermented food or dietary supplements that intends to give health benefits when consumed or applied to the body. They are not harmful microbes/germs but instead are helpful in digesting food and destructing disease-causing cells. They are almost similar to the microbes that are naturally present in the intestinal tract of human bodies.


Probiotics contain a variety of microorganisms. The most common are lactobacillus and bifidobacterium species. Other species are also present that are widely used in industries like marine biotechnology and cosmetics as well as probiotics can be used in various dosage forms formulated in paste, creams, oral capsules, tablets, in curd, yoghurt, cheese preparations, and strength is usually measured as million/billion colony-forming units (CFUs) per capsule.


lactobacillus species

bifidobacterium species

other species

L. acidophilus

B. bifidum

Streptococcus thermophilis

L. casei

B. longum

Enterococcus faecalis

L. reutri

B. breve

Escherichia coli

L. bulgaricus

B. infantis

Bacillus cereus

L. plantarum

B. lactis

L. johnsonni

B. adolescentis

L. lactis

Some people confuse with similar and common terminologies like pre-biotic or syn-biotic which are different from probiotics. Pre-biotic are food components that tend not to digest and they stimulate the growth of microbes already present in the gut. Where synbiotic are mixture of both probiotics and prebiotics.

Probiotics are beneficial as they guard against diseases common to infants and gastro-intestinal tracts of humans and animals. Also, marine life like fishes and shrimps are dosed with probiotics for healthy benefits. The most common diseases that are treated by probiotics are:

  • Antibiotic-associated diarrheas

  • Sepsis in infants

  • Periodontal disease

  • Infant colic

  • Constipation

  • Inflammatory bowel syndrome

  • Hay fever

  • Asthma

  • UTI

  • Upper respiratory infections and many more problems

They also help the body in maintaining and influencing immune response. People who used to think science as in anti-bio, because health was generally categorized as “drug-like” as drugs are commonly used for preventive and therapeutic purposes, now have a different views after these drugs are replaced by probiotics (quite frequently used as drug replacements).


Steps to consider before scaling up Producing quality of fermented products is both art and science. Even if quality products are produced, it is a real challenge to maintain consistency of the quality probiotic products. The overall process should be standardized as the production level is indifferent to small scale, dried and suspended in buffer lab-scale microbes may not necessarily produce at same growth in a larger scale. We will see the factors which are necessary to be looked upon while going from lab to larger scale in this blog!

  • Pre-market registration

Just like drugs require clinical trials for efficacy, probiotics need pre-marketing authorization prior to their use. It is a process of assessment based on their quality, stability, viability and reliability. This is a global concern but, in few countries like India, it is not a concern of efficacy. Some industries directly produce probiotics without pre-authorization. Actually, if we compare drugs to probiotics, drugs have more therapeutic and preventive purposes than feed supplements/ probiotics. That is why drug registration is more important than just a feed supplement but, there are few registered probiotics in India that are used for medicinal purposes. Unlike India, there are few countries who strictly follow registration method and have guidelines for this particular process (eg. Europe). This is an advance checkpoint where everything is into consideration and it is an achievement for European government for providing such crucial steps towards health.

  • Evaluation at smaller level


The foremost step before going into scale-up of probiotics is to have a thorough knowledge and understanding of physiological and biological effects of probiotics on humans and animals. It is necessary to know the mechanism of intestinal microbiota, the type of strain to be used, the environment of GIT and mode of product delivery. Some promising strains are already present in the market that are safe to use widely in industrial feed applications. It is essential for a strain to be potent and safe. Many organizations like FDA, GRAS, EFSA have listed guidelines on safe strains for application use that are not only safe at global concern but also for people who are working on it. There are number of researches on microbial species but very few research about their safety to humans and its ability to thrive in host environment. When we say safety, we mean that strain should be devoid of toxins, allergy or irritation reaction and other virulence factors to the person who will handle the process. For probiotic scale up microorganisms should have efficiency, adaptable properties, optimized industrial productivity, authorization of their use and ability to thrive in host.

Only a selective microbe provides desired benefits hence not all probiotics work in similar way so, selection should be based upon the required activity. Everything depends on the viability, stability and activity of microbes. Any change of growth condition affect microbes’ behavior, it is important that its purity and activity is continuously tested at every checkpoint by accurate testing procedures.

  • Optimization

A continuous research and development is required to optimize fermentation as well as down streaming of the probiotic process. Process usually starts with a few microliters of suspension from a cell bank vial inoculated into inoculation media and cultured overnight in a shaker incubator or without shaking (depending on the culture). Next day, this specific inoculum is added to a big flask containing media for culturing and further it is added to production media in big fermenter for higher growth. At their optimal growth conditions, microbes multiply and are transferred to large vessels. Downstream is the major part where product is filtered through filtration process which is further concentrated and lyophilized/ spray dried to powdered form. Quality of the product is tested from QC department which leads to packaging and manufacturing. This process is not as easy as it looks like. It takes a whole lot of work and determination with speed and accuracy simultaneously. Each step is a checkpoint where tests should execute for proper product recovery like pH, temperature, pressure, etc.

Following are some elaborated points on the above checkpoints to be looked upon deeply and ethically.

  • Microbes generally start with lag phase, heading to log phase (maximum growth) stationary phase and death phase when they are multiplying. So, the process usually lags behind due to fermentation halting and immediate action is required during that specific time or phase where it is going exponential level to gain maximum yield. May be adding feed, changing pH or temperature will do the same.

  • Cryoprotectants usually used during lypholization step effects viability of microbes. Thus, this parameter should never be underestimated. For this reason, sometimes dry spray is suggested, an alternative that have shown some promising effect on microbe stability.


(Fig. Showing spray drying in development of Probiotics)

  • When Packaging and manufacturing happens, there is a need to check micro reliability and sustainability since probiotics are packaged by grinding, mixing, blending, compression and in case of tablet, homogenizer and granulation methods, which are mechanically very rough for microbes to handle.

  • Humidity levels in packaging area, packaging material, temperature of the room affects microbe survival rate.

  • Also, since there are different dosage forms, material should be handled accordingly and wisely. For example, in case of solutions, suspensions and blends- wetting agents, buffering system, salt formation ingredients, coloring and flocculating agents, sweeteners and flavors should be used only when tried at smaller scale and should not disturb strain's shelf life.

  • Inducing heat tolerance, altering temperature during growing phase, harvesting at proper stage, altering pH at defined phase may influence whole process in a good way.

  • More often people use spore forming bacteria as a culture as they survive higher temperatures, vigorous centrifugation, resist harsh conditions thus report good survival rate.

  • Entry room in a plant should have air locks, HEPA filters, ULPA filters, HVAC and AHU for conditioned atmosphere causing loss of contamination chances.

  • Initial frozen stock of cells should be free of contamination i.e. only one colony cells as single pure strain.

  • Fermenter should be of good quality and provided with every facility like fermenter from Amerging Technologies. Manufacturer of fermenters with good quality stainless steel, fully automated, with all kinds of pH, temperature and pressure controls.

  • Usually after filtration, product is still diluted and contains impurities, which can be filtered by TFF method. This method not only concentrates the product but also filter down salts and viral particles.

  • Other major checkpoint is clean in place and sterilization in place (CIP/SIP). It is a good method to wash, clean and sterilize large fermenters without dismantling and transport. But, sometimes cleaning residues reside in vessel and later on hinder fermentation process.

  • Manufacturing of the final product (dietary supplement) needs to happen under strict temperature- and humidity-controlled conditions. Establishing a low-water-activity product starts with sourcing dry carriers, excipients, and other active ingredients that will be blended with the probiotic. Small amounts of high-water-activity ingredients can be added as long as the total water activity remains below 0.2 or, ideally for long shelf lives, below 0.15. Once the low-water-activity probiotic format has been produced, those conditions can be maintained by choosing packaging with adequate moisture vapor transmission rates (MVTR).

  • Not all plastic bottles are equal in terms of sustaining probiotic viability. Polyethylene terephthalate (PET) bottles should never be used, as their structure allows migration of too much moisture compared with high-density polyethylene (HDPE). While glass bottles have the best MVTR, a bottle seal that adheres well to glass must be chosen to prevent moisture ingress from the bottle opening.

By adopting good laboratory practices (GLP), the QC lab minimizes cross contamination. This includes personal hygiene, the proper personal protective equipment (PPE), establishing foot traffic protocols, product flow through the lab, and sanitation procedures and logs. Hazard analysis and critical control points (HACCP) is used to identify critical control points and establish acceptable protocols to minimize hazards.



#probiotics #scaleup #labscale #pilotscale #productionscale #biotechnology



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