Production of probiotics pdf

Probiotics, particularly when included in dietary supplements, are commonly transported and stored at ambient temperatures and humidity.

In order to provide the target dose until end of shelf life and to compensate for potential losses during storage and handling, an overage is commonly included in the product [ 7 ]. There are many articles available on the identification of potential new probiotics and their safety [ 8 ], as well as about the health benefits of specific probiotic strains or strain combinations [ 9 ]; however, these topics are not discussed here. Also, for the market potential and health-economics of probiotics, the reader is referred to elsewhere e.

Here, we discuss what is required to reliably and reproducibly produce high-quality, safe, and stable probiotics and what it takes to keep them alive in sufficient numbers in various delivery formats in order to provide efficacious probiotics and their health benefits to the consumer.

LAB and bifidobacteria are commercially manufactured to satisfy customer demand for probiotic dietary supplements and dairy starter cultures.

From a manufacturing standpoint, the desired commercial product will have as high a yield as possible and consist of viable, concentrated cells that are stable and will have consistent performance in the intended application. High cell count and long shelf-life stability in a variety of different temperature and humidity conditions are expected by customers, especially for high-quality dietary supplement products with doses established through clinical trials.

In contrast, rapid and consistent acidifying activity in milk is desired by customers for dairy starter cultures. In this section, the manufacturing process is briefly described and important challenges are highlighted for manufacturing and consistent product performance.

The manufacturing processes of LAB and bifidobacteria for dietary supplements and dairy applications have the following steps in common, as shown in Figure 1. Frozen seed stock, which has been carefully prepared to consist of a single pure strain and verified to be free of contaminants by quality control QC testing, is used in a limited number of sequential seed fermentations to achieve the desired inoculum volume and is ultimately transferred to the main fermentation vessel for growth.

Alternatively, frozen direct vat inoculation DVI material consists of a larger amount of concentrated cells that can be used to directly inoculate the main fermentation vessel. The aim of both approaches is to limit the number of generations from seed stock to product, thereby reducing any potential risk for genetic drift.

The heat-treated medium used in the seed scale up and main fermentation is a blend of water, nitrogen sources, carbohydrates, salts, and micronutrients necessary for growth. The fermentations are carefully controlled and after the fermentation in the main tank is completed, the cells are concentrated by separating the cells from spent medium through centrifugation. Depending upon the final product application, stabilizer solutions i. Cryoprotectants inhibit the rate of ice growth via increasing the solution viscosity and keeping the amorphous structure of ice in close proximity of the cell.

Lyoprotectants stabilize the lipid bilayer structure of the cell membrane in the absence of water [ 12 ]. Commonly used cryo- and lyoprotectants are carbohydrates and peptides. In the dairy industry, skim milk powder is often used [ 13 ]. Once the probiotic concentrate is blended with the cryoprotectant solution, various freezing processes can be applied. One simple freezing technique consists of pouring cryoprotected concentrate into cans and immersing the sealed cans into a liquid nitrogen bath.

The frozen cans can then be shipped to companies incorporating probiotics in food or beverages. Alternatively, a more productive technique consists of pelletizing the cryoprotected concentrate by dripping the concentrate through calibrated holes into a bath of liquid nitrogen.

Alternatively, frozen cell pellets can be used for freeze-drying lyophilization to a dried end-product. The frozen pellets are transferred onto trays which are placed on top of shelves.

The shelves have the capability of being temperature controlled and are progressively heated once vacuum is established in the freeze-drying chamber.

An alternative option consists of filling trays with the cryoprotected concentrate. The trays are then placed on top of temperature-controlled shelves which are initially cooled down to freezing temperatures under atmospheric pressure.

Once the concentrate in each tray is frozen, the shelves are gradually heated once vacuum is applied. Freeze-drying length varies as a function of the strain, its formulation, and the freeze-drying cycle but usually takes a few days to be completed. Schematic representation of the production of probiotics for dietary supplements and dairy starter culture strains. After removal from the dryer, the lyophilized material is milled to a powder with a defined particle size and density.

The milled material can then be used for blending with excipients bulking agents , additional functional ingredients if required, and flow aids, depending on the needs of the customer.

The blend is then used to make finished formats such as capsules, sachets, or tablets. QC testing is performed on in-process samples and the final product to make sure that the end-product is high quality and free of contamination.

During development work, special care is taken to understand the production conditions involved in manufacturing probiotics at a commercial scale and to evaluate the performance of strains under similar conditions at lab scale.

Each step in the process depends upon the prior step and it is important to identify strain-dependent sensitivities and to try to maintain the overall health of the cells while proceeding through the process. Scale up can sometimes be very challenging because the down-sized process for making cells during lab-scale development work is inherently more tightly controlled and has shorter hold times during each step in the process.

For example, commercial separation of cells from spent media by centrifugation may take hours because of the large volume of cells compared to minutes at lab scale with the smaller volumes involved; this leads to different stresses i. Also, there are multiple steps where cells are pumped during commercial-scale production which do not typically happen during bench-scale development work.

In addition, cells during commercial production are likely to experience different pH and temperature conditions that are not easily reproduced in the same way at lab scale. Hence, the importance of scaling up to an intermediate volume in pilot, so that these more representative production conditions and stresses can be evaluated and mitigated before proceeding to commercial production.

Scale up from pilot to commercial scale maybe challenging for the same reasons as scaling up from lab scale to pilot. As mentioned previously, the hold times at various steps in the production process can greatly exceed those at lab scale. In order to ensure that the cells are robust and will meet shelf-life claims, the cells manufactured in pilot- and commercial-scale production are evaluated for several hours past the typical hold time that they would normally encounter at the different steps in the process.

If adequate robustness is not demonstrated and an adjustment cannot be easily made to mitigate this sensitivity, the strain will return to the laboratory for another iteration of development work before being scaled up again. Generally, if a strain seems to be especially sensitive and difficult to develop at lab scale because of strain-dependent sensitivities, it is highly likely that additional challenges will be encountered during scale up to pilot and subsequent scale up to commercial production.

The same is true for the robustness of the cells with the hold times involved in the production process. Often, there are additional iterations where the strain will return to the laboratory for additional development work to try to redevelop and overcome these identified sensitivities and robustness issues before scaling up again.

Like fermentation, freeze-drying must be evaluated at the bench-scale level before commercial production. An optimal freeze-drying cycle can be defined through an iterative process where pressure, heating plate temperatures, and frozen pellet bed thickness are adjusted until appropriate water activity Aw , cell count, and shelf-life stability are achieved [ 16 ].

In addition, cryoprotectant formula or dosage can be adjusted if cell survival is not satisfactory after the iterative process [ 17 ]. It is imperative to determine if the lab freeze-drier operation can be scaled up to an industrial freeze drier. In particular, the condenser capacity, condensing rate, and heat transfer of the industrial freeze drier must be known and sufficient to eliminate the moisture released during the drying cycle. LAB and bifidobacteria are fastidious microorganisms in terms of nutritional requirements for growth and performance.

LAB and bifidobacteria tend to be auxotrophic for some of the 20 amino acids and have nutrient requirements that need to be satisfied from the external environment to grow. The complexity of these auxotrophies and nutrient requirements is often linked to the nutritional potential of the environment to which the microorganism was adapted and from which it was sourced [ 18 ].

For example, Lactobacillus plantarum sourced from plant material has fewer auxotrophies [ 19 ] and more biosynthetic self-sufficiency than Lactobacillus johnsonii isolated from the upper gastrointestinal tract of a human being, which is an environment with a greater availability of nutrients such as free amino acids, short peptides, and oligosaccharides [ 20 ].

Identifying strain-dependent nutritional needs requires a multidisciplinary approach that is both knowledge and empirically based. The power of evaluating the genome i. Also, analyzing the sterilized medium prior to inoculation and after fermentation i. Using these approaches to also understand the composition of complex raw ingredients, yeast extracts, yeast peptones, milk, and other complex nitrogen sources helps match critical fermentation media ingredients with the nutritional needs of the strain under development and provides an opportunity to adjust the medium and process to achieve better strain performance and better manage manufacturing costs and efficiency.

Finally, there is a wealth of empirical data that can be collected that is not easily obtained or even predicted using the more knowledge-based -omics approaches. With the right expertise and innovative philosophy, these approaches, when combined, are extremely powerful for understanding strain dependencies, strain sensitivities, nutritional needs, and nutritional limitations, so that high-performance strains can be successfully developed and manufactured that satisfy customer needs.

Given the importance of the fermentation medium for manufacturing LAB and bifidobacteria, changes to the raw materials can have a profound effect on growth and performance. The changes to raw materials by the supplier could be due to cost savings with process improvements, a change in ingredient sourcing, and variation within the manufacturing process.

Such changes to complex ingredients such as protein sources e. Depending on the nutritional requirements and sensitivities of the strains being manufactured, the lot-to-lot variation in complex raw ingredients can sometimes go undetected, with some strains having seemingly consistent performance, whereas the performance of other strains is more obviously affected in a positive or negative way.

With more complex ingredients such as yeast extracts, the differences responsible for the change in strain performance may not be readily attributed to the amino acid, peptide size distribution, vitamin, nucleotide, salt, and carbohydrate levels but rather due to the presence or absence of some other unknown or less obvious components.

Carryover of components used to culture the yeast to make the yeast extracts and peptones can have no obvious effect on strain performance or can positively or negatively affect the performance of probiotic strains in a strain-dependent manner. Also, cane and beet molasses can be sourced from all over the world with performance and quality which can have lasting effects when carried over into fermentations with yeast extracts and peptones [ 25 ].

Manufacturing LAB and bifidobacteria with consistently high performance is also dependent upon how well controlled the manufacturing process is. Unsurprisingly, there is considerable diversity between strains, even from the same species, in terms of sensitivity and response to the manufacturing process, which affects performance [ 26 , 27 ].

During lab-scale development, scale up to pilot, and subsequent commercial scale up, these sensitivities are discovered and the process is adjusted so that consistent high performance can be achieved. Once the process for each strain is established, it is important to run this process the same way each time. Controlling the manufacturing process occurs at several levels that include the following:. The raw material suppliers are audited and raw materials evaluated at some level to ensure high quality.

Establishing meaningful and achievable ranges for process parameters and verifying process capability for consistently being within those ranges. Automating the process as much as possible to reduce inconsistency associated with human error and manually controlled aspects of the manufacturing process.

Evaluating captured data from the process and using Six Sigma approaches [ 28 ] for continuous improvement and making sure the process is being reproduced as consistently as possible. Evaluation of in-process samples and final product to ensure the product is of high performance and free of contaminants.

The reality of the manufacturing environment is that there will be lot-to-lot variation in raw materials that will not be discernible until they are used in manufacturing. The production plant environment is dynamic, with new equipment installations and new processes being implemented that can perturb plant steady-state operation for a time.

Also, some aspects of the manufacturing process are going to be more manual and less automated. There will be shift changes for operators and employee turnover.

Equipment can and will unexpectedly fail. Probes and sensors used to monitor different steps in the manufacturing process can malfunction. Each of these examples are challenges that can affect the performance of the strains being manufactured, because in these circumstances, the process conditions and hold times are different and perhaps outside the range explored during strain development work and scale up.

Manufacturing experience has demonstrated that process differences resulting from examples such as these, even seemingly minor and superficially unimportant, can have an outsized impact on performance that is usually, but not always, negative for performance. Even strains considered to be well understood and reliably manufactured can have surprisingly poor performance if the change or difference in the process is outside what was established during development, scale up, and implementation.

Sometimes, efforts to troubleshoot and correct the process issue is confounded because the differences between production runs is multivariate and not readily identifiable. This suggests that some aspects of the process are not adequately monitored and controlled to ensure consistently high strain performance. The importance of controlling the process cannot be understated for consistently manufacturing high-performance LAB and bifidobacteria, especially given the strain-specific nutritional requirements and process sensitivities, as well as subsequent cell responses to the process steps that affect performance.

In order to manufacture high-performance probiotics and dairy starters, the unique nutritional requirements and sensitivities of different aspects of the manufacturing process of each strain have to be well understood and accommodated within the manufacturing process. These strain dependencies are typically identified and worked out during lab-scale development, scaled up in pilot, and scaled to the commercial level before the process is finalized and commercial manufacturing proceeds.

Developing a tailor-made manufacturing process to accommodate these strain dependencies poses additional challenges because of the complexity associated with sourcing and managing raw materials, as well as managing numerous manufacturing processes in the production facility. Fortunately, the strain dependencies associated with mesophilic e. Conversely, the complexity of the manufacturing processes for probiotics destined for dietary supplements is considerably greater because of the ever-increasing number of probiotic species in demand by customers and the diversity of species and strain dependencies that are involved.

To successfully manufacture high-performance probiotics and dairy starters, the strains need to be well understood in terms of nutritional needs [ 18 , 19 ] and process sensitivities [ 8 ]. Also, the composition of complex raw materials i. The manufacturing process has to be very well controlled, so the strain can be consistently made with subsequent predictable performance.

Finally, the organization has to be willing and able to manage the high degree of complexity incurred through the ever-increasing number of raw materials and tailor-made processes necessary for manufacturing high-performance dairy starters and probiotics.

As outlined above, raw materials for the production of probiotics and dairy starter cultures need to be carefully selected and controlled.

These may relate to Kosher and Halal requirements but may also concern the absence of certain allergens from the final product. This maybe challenging, as some of the commonly used media for the culturing of microorganisms in general and LAB and bifidobacteria in particular would not comply with these requirements. For Kosher, this means Kosher ingredients, no mixing of dairy and meat products, and the use of Kosher methods.

For allergens, avoiding the most common dietary allergens, such as dairy, soy, gluten, and nuts. For vegetarian and vegan production, meat and dairy sources have to be avoided, respectively.

For dairy starter cultures, obviously a dairy-based medium can be used. For dietary supplements, the omission of dairy, soy, and meat extracts requires searching for alternative nitrogen sources. Carbon sources derived from wheat have to be avoided because of potential contamination with gluten. Quality control and quality assurance both have the same goal of producing a quality product for sale, but they differ in their approach.

Quality assurance is responsible for maintaining quality systems within the facility so that product defects and mistakes can be kept to a minimum.

Quality control is responsible for the actual testing of raw materials, in-process samples, intermediate samples, and end-product samples, which can involve a wide variety of examinations.

Before end-product testing can occur and the results considered sufficiently reliable to be added to a certificate of analysis COA , the QC lab needs to have implemented several programs which help to ensure the quality of the product. As ever-more probiotics are being consumed by the general population, which includes vulnerable subjects such as pregnant women, infants, people with allergic reactions, and those with compromised immune systems, the standards and rules for maintaining QC labs and testing have advanced [ 29 ].

Compliance with regulatory guidance and applicable standards have improved the control and outcome of production and QC processes, augmenting the ability of production facilities to produce and QC to release a more consistent end-product. Examples of regulatory guidance and applicable standards that are being embraced are given in Table 2. Additionally, by adopting good laboratory practices GLP , the QC lab minimizes cross contamination by understanding the steps used in production to make the end-product, the composition of the end-product, and the handling of the end-product.

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.

Utilizing customer as well as in-house auditing of the QC lab attempts to identify continued areas of concern and to make sure that methods are being followed. Metrology is equally as important as GLP in establishing a robust program for the qualification and calibration of lab equipment. Programs need to be set in place to make sure that the equipment, such as autoclaves, incubators, clean room environments, pipettes, etc.

Metrology records are also created, maintained, and retained. Also included is the monitoring of the air handling and water quality, which is important in preventing any contamination downstream.

Every lot of material which is produced should be associated with a batch record which includes all production and QC paperwork. That file should then be reviewed by quality assurance to make sure all relevant paperwork is present and regulatory compliance is met. The length of time which all batch records, metrology records, etc. To avoid quality issues being caused by inferior raw materials and packaging, vendors and their raw materials should be qualified for use ahead of time.

This means that the following information should be known and approved by the QC team before use:. GMO status; allergen status; the raw material purchasing specifications, including chemical, physical, and microbiological; food grade quality; pesticides; irradiation; Kosher rating; raw material packaging type and size; storage condition; shelf life; and the safety data sheet SDS should be reviewed. It is also helpful to review a COA from the vendor.

It should also be determined what type of inspection needs to occur once an approved raw material arrives at the plant. This can include appearance; identification; chemical, physical, and microbiological; review of the vendor COA; identification that the material is proven to be what it is; and the frequency of the QC checks.

For example, does a QC of the raw and packaging materials need to happen with every new batch, or can a skip program be established? The trained skills of the laboratory technicians should be monitored by establishing a program whereby the accuracy of quality tests can be checked against known controls. Retaining skilled laboratory technicians and maintaining their skills will help minimize inconsistency among results. Information which needs to be considered in order to create a representative sampling plan is: how many samples should be taken which accurately represent the batch?

Statistical programs have been developed, which take into consideration the sample s , that provide the reassurance that the batch is acceptable while avoiding over-testing, which saves both technician time and money. Additionally, if more than one sample is taken, or if production runs multiple sublots within a batch, do they need to be run separately or can samples be consolidated? Sample size also needs to be considered. Retained samples from each production lot should be stored at the same recommended temperature supplied to the customer.

The sample size should reflect material taken over the production run and accommodate full QC testing. In general, all of these events involve the steps indicated in Table 3. The creation of release specifications needs to include customer requests, the capability of production, and the absence of pathogens.

With the support systems in place, questions concerning the type of end-product testing required need to be addressed. Instead, the customer directly ingests the bacteria. What type of environment surrounds the bacteria to be sold to the consumer? That is, are the bacteria in a freeze-dried form that will be packaged in capsules, sachets, straws, etc.

What type of consumer is being targeted by the customer? Senior citizen? Examples of end-product testing are listed in Table 4.

Future concerns are efficiency and automation. A need for future methods that can be validated and accepted by the customer is in definite need. Additionally, customers want more accurate identification, especially among bacterial blends.

The inclusion of probiotics in dietary supplements primarily utilizes probiotics in the freeze-dried powder format. Capsules, tablets, and powder in stick packaging or sachets are the most commonly found formats on store shelves and are usually stored at ambient conditions.

Dietary supplement products should deliver the probiotic count declared on the label throughout the shelf life of the product. This ensures that the consumer receives the adequate dose of probiotics to affect the targeted structure function health claim or the otherwise suggested health benefit.

It is important to characterize the stability of each strain so that the proper amount of overage can be added during the production of the dietary supplement format to ensure minimal counts of each component strain in a multi-strain formulation.

However, the quality control of single strains in a multi-strain formulation is very challenging, especially with multiple strains from the same species. Currently, no generally applicable and reliable methods exist, although experimental molecular-based techniques have been explored [ 30 ]. Dietary supplement formats typically have shelf lives measured in years, so a great deal of care must be taken to make sure that the proper probiotic cell count is maintained.

Because probiotics, even in the freeze-dried state, are live microorganisms, more consideration must be given to their handling and storage than for other dietary supplements and food ingredients [ 16 , 31 ]. Water activity, followed closely by storage temperature, is the main factor impacting probiotic stability over the shelf life of the product [ 32 ]. Downstream processing and 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. The relative humidity of the processing facility must also be kept low so that the probiotic dietary supplement does not absorb moisture from the environment during production.

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 Table 5. The lower the MVTR, the slower the moisture ingress. Preventing moisture ingress helps to maintain a viable probiotic cell count over the shelf life of the product. 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.

The addition of desiccant packs into the bottles aids in maintaining the low water activity of the probiotic contents. Chewable tablets and mixing probiotics with other active ingredients are two trends currently seen with commercially available dietary supplements. These two products introduce additional variables which may impact probiotic count and survival.

Compression during tableting tends to reduce the viability of probiotics in the tablet [ 33 ], and other active ingredients can contribute to an increase in water activity as discussed above. Also, ingredients of plant origin may be rich in polyphenols, which may be antimicrobial [ 34 ]. The compression pressure used in chewable tablet production can decrease the probiotic cell count dramatically. The extent of the compression cell count loss will depend on the formulation and probiotic strain [ 35 ].

Tests should be performed to understand the minimum amount of compression required to produce a tablet with acceptable friability characteristics in an effort to preserve as many live cells as possible. As mentioned above, ingredients in the chewable tablet formulation should be chosen with the lowest possible water activity. When considering other active ingredients to be mixed with probiotics, there are generally two stages that should be evaluated.

In the dietary supplement format, it will typically be the water activity of the other active ingredient s that can be detrimental to probiotic survival if they increase water activity. The second consideration is the impact of the active ingredient s on the freeze-dried probiotics upon rehydration, which occurs after ingestion in the stomach or when a buffer solution is added before enumeration of the product in the laboratory [ 16 ].

The exact impact of the active ingredient s on the probiotic in vivo can be difficult to determine because of the logistics required to sample from a person. Gastric simulators can be employed for an approximation of the in vivo system, but then careful consideration should be taken regarding simulator set up. For interaction of the active ingredient s with the freeze-dried probiotic during the enumeration process, an interaction assay should be first conducted to determine if the ingredient decreases the probiotic count upon contact.

The interaction assay could consist of enumerating a rehydrated sample before and after a min hold time. If the cell count is reduced after 30 min, mitigation steps in the enumeration procedure, such as making a bigger initial dilution e. Consuming probiotics in a food may be perceived as a more natural way of receiving a daily dose [ 36 ]. The shelf life of probiotic nondairy foods is generally shorter than that of dietary supplements, but the matrices can be harsher on probiotic survival.

When adding probiotics to juice, there are many factors to consider: pH, acids, anthocyanins, and the fact that the probiotic is in a vegetative rather than a freeze-dried state, as in dietary supplements [ 37 , 38 ]. Refrigeration of the juice is required to help maintain probiotic viability so that the adequate dose is delivered throughout shelf life and also to avoid metabolic activity of the probiotic and spoiling of the juice [ 39 ].

Secondary packaging options enable the production of shelf-stable juice, where the probiotic is in a separate compartment e. There are many food formats that can successfully incorporate probiotics and deliver them at the required dose and usually involve experimentation to find the optimal formulation and strain combination. Chocolate, crackers, breakfast cereal, snacks, chips, peanut butter, and crispy granola bars are among the many options for probiotic foods.

The main factor impacting probiotic stability in these food formats is water activity. Generally, the water activity must not be more than 0. The exception to this water activity guideline is fat-based foods such as chocolate and peanut butter, where good probiotic stability can be achieved despite higher water activities up to 0.

Possibly the best-known traditional probiotic foods, yogurts and similar fermented milk products, have a strong association with gut health in many societies globally. Fermented milk products stand out as a special type of matrix for carrying probiotic health benefits, in that they offer the possibility of increasing the probiotic population during the fermentation step, thus providing cost-efficient cell counts. The downside, however, is that care must be taken to assure that growth of an added probiotic culture does not compromise the sensory profile of the product.

Thus, in order to benefit from this, extra diligence must be given to quality assurance during prototype development sensory aspects as well as during commercial production, where the clinically relevant dose must be reliably met during manufacturing.

Furthermore, if not managed carefully, the fermentation step imposes a potential health risk, in that pathogens may be allowed to grow alongside probiotic and starter cultures. Such a risk can be mitigated by applying dedicated and strict hygiene standards for fermented products. The probiotic genera Lactobacillus and Bifidobacterium , which have a long tradition as starter cultures in yogurt production, generally exhibit good survivability in fermented milks, although very-low-pH products can be too acidic for certain strains [ 40 ].

Thus, finding the right balance between clinical dose, shelf life, and cost efficiency will in many cases be a function of strain type and pH. Other parameters that influence successful incorporation of probiotics include fermentation temperature affecting probiotic growth , storage temperature, packaging type oxygen transmissibility , processing steps such as heat treatment and homogenization, and interaction with other ingredients [ 41 , 42 ].

As the addition of fruits and increasingly grains is commonplace in yogurt production, it should be stressed that certain types of fruits and grains can be particularly detrimental to probiotic survivability [ 43 ].

Therefore, prescreening the compatibility of a given probiotic strain with select ingredients of interest is a recommended approach to prototype development. Pellets will more conveniently allow homogeneity of the product and reduce mixing time, whereas powdered probiotics can lead to issues with wettability and dispersibility. Ice cream poses another interesting yet different matrix for carrying probiotics. In contrast to fermented dairy, where probiotic viability is measured in weeks or, in optimal cases, months, ice cream is able to accommodate probiotic strains at clinical levels for more than a year [ 44 ].

Other factors typically associated with ice cream which aid probiotic stability include neutral or closer to neutral pH, high total solids, and, especially, fat content. There are, however, obstacles to overcome before successful inclusion of probiotics is achieved in ice cream products.

Overrun, which aerates the ice cream mix prior to packaging, may cause a drop in viable cell counts due to increased oxygen incorporation. This can be compensated for by adding overage or by culturing the probiotic population during a potential fermentation step prior to the actual ice cream manufacture.

Additionally, the toxic activity of oxygen might be accommodated by utilizing aerotolerant species such as lactobacilli, rather than strictly anaerobic species. Another special challenge associated with probiotic ice cream production is the freezing step, which can compromise the bacterial cell envelope and thus impose a decrease in cell count in its own right [ 45 ].

In order to minimize this threat, it may be advisable to employ a rapid freezing step to control ice crystal formation in both the product and the bacterial cells within. Although probiotics in cheese are not widespread, cheese is very well suited as a carrier and many examples of successful inclusion have already been documented [ 46 ].

Most cheeses have a high fat content, often combined with a relatively low water content. When further combined with cool storage conditions, this allows for probiotic viability at clinically relevant levels after several months of shelf life. The propensity for growing probiotic cell counts during the ripening process in cheese-making even makes it feasible to include a typical daily clinical dose of probiotics in very small amounts of cheese.

Thus, a mere serving of 10 g of cheese would be sufficient to obtain the desired daily dose of 10 9 CFU [ 49 ]. For all media, pH was adjusted to 5. Of these, glucose, corn steep liquors and sodium nitrates were studied for their effect on cell mass production.

Different set of experiments were conducted for cultivating cells in different ratios of these three main nutrients. Optimization of key nutrients concentrations in Shake Flask The medium 2 was selected to undergo for optimization process. For optimization experiments, the key nutrients were being identified and prepared at different concentration for three separate experiments. Glucose was prepared and autoclaved separately and added to other medium components before inoculation.

Bioreactor Cultivation For bioreactor cultures, cultivations were carried out in pilot scale L stirred tank bioreactor bioreactors of 8. Agitation was set at RPM and conducted by two, 4- bladded Rushton turbines. For all cultures, the pH was initially adjusted to 5. The bioreactor was equipped with pH probe, oxygen probe, foam sensor.

Analysis Sample preparation, biomass and ethanol determination Sample in form of 2 flasks of 50 ml fermentation broth each or 20 ml in case of bioreactor cultivations were taken at different time intervals during cultivation process.

The yeast biomass was then calculated based on previously prepared standard curve, in which one unit of OD was equal to 0. Samples were then centrifuged immediately at RPM in 50 mL falcon tube. The cell free supernatant was then used for ethanol determination. The GC was equipped with an internal air compressor and hydrogen generator. N2 was used as carrier gas with pressure control 14 PSI. Syringe was thoroughly washed with ethyl acetate between injections to avoid cross- contamination.

Chromatography software from Perkin Elmer Turbochrom 4, ver. As shown in Figure 1 different media supported cell growth and alcohol production by different extent. Media 1, 2 and 4 included glucose as carbon source while media 3 and 5 have sucrose and lactose as carbon source, respectively.

The highest cell mass of about 2. Medium 2 include The higher amount of glucose can supported high biomass, production of organic acids in small amount and thus lower the pH as consequently. However, it has been also observed that this medium produce alcohol in lower quantities compared to other cultures except medium 4 which produce very low cell mass and low alcohol as well. Medium which produced high amount of alcohol is unfavorable as this will be on the cost of aerobic yeast growth and biomass production.

This resulted in significant reduction of cell growth rate with increasing in ethanol production rate [18,19]. In this screening, medium No. The pH for all media was lower than the initial value of pH 5. Based on these highest biomass productions, medium 2 was selected for biomass production. Kinetics of cell growth and alcohol production by S. Cell growth, alcohol production and change in culture pH were followed every hour. Figure 2 showed the typical growth kinetics of Saccharomyces boulardii for 24 hours cultivation.

As shown in Figure 2, cells grew exponentially with specific growth rate of 0. Cells grew thereafter with very low rate until the end of cultivations. This was due to the cells entered stationary phase after 12 hours where the cell growth was limited by the concentration of substrate and accumulation of alcohol.

Its also worthy to note that, during the active growth phase the first 5 hours , the pH of culture dropped gradually reaching 4. On the other hand, alcohol was accumulated in culture throughout cultivation time and the maximal alcohol production of about 0. Thus we can conclude that, the growth was limited after 12 hours cultivations and the rest of cultivation time was mainly for alcohol production.

Effect of different glucose concentration on S. As shown in Figure 3, the biomass increased with the increasing of the initial glucose concentration in culture in the range between 0 and 20 g L The maximal value of obtained biomass was 3. Beyond this concentration, the biomass decreased slowly with the increase of glucose concentration up to g L This showed that after 20 g L-1, higher glucose concentrations did not exhibit any significantly improvement in biomass production of S.

On the other hand, the alcohol production increased when the concentration of glucose increased even under aerobic cultivation condition. High glucose concentration supported ethanol formation, which indicates metabolic regulation not only by oxygen, but also by glucose through crabtree effect [21].

While the pH for cultivation in different glucose concentration was in a range between pH 4. Therefore, we can conclude that the best concentration of glucose for S. The decrease in biomass production and the increase in ethanol production beyond this concentration was due to the crabtree effect [22].

It have been reported that medium supplementation with nitrogen sources such as ammonia, glutamine and asparagin supports higher biomass production during yeast cultivation compared to other sources such as urea and proline [23]. In this study it was clear that addition of corn steep liquor as nitrogen source to medium promoted cell growth of S.

Corn steep liquor is widely used in fermentation medium and not only acts as nitrogen source but also considered as potential source for growth factors, amino acids and salts [9].

In addition, use of low price substrate such as corn steep liquor helps also to reduce the overall production cost espceially in large scale production. In this experiment, different concentrations of corn steep liquor CSL ranging from 0. As shown in Figure 4, the maximal biomass production of about 3.

However, increasing the CSL concentration from Furthermore, it was observed that the addition of CSL in concentration more than 30 g L-1 of CSL decreased further the cell mass, of 2. The maximal value of alcohol production of 0. It is also worthy to note that, the pH was dropped from value of 5. In conclusion, the best concentration supported of yeast probiotic of S. Other study by Spigno et al. In others study done by Kim and his group CSL was also supportive organic nitrogen source for cell growth of S.

Sodium nitrate is considered as important inorganic nitrogen source as it was previously reported for its positive role in the biological synthesis of single cell protein and invertase enzyme production during S. In this study, it was observed that the addition of sodium nitrate in concentration of 1. The lowest biomass was about 2. However, the addition of sodium nitrate from 2. In general, from the results in figure 5, addition of different concentrations sodium nitrate from 2. On the other hand, the highest alcohol percentage reached about 0.

However, increasing the sodium nitrate concentration 5. For the pH change in culture, all the values were almost the same and in the range of pH 4. The drop of pH was due to formation of alcohol and other by-products, such as organic acid by S.

From this study it was concluded that the best concentration of sodium nitrate for yeast biomass production is 1. As shown in Figure 6, cells grew exponentially without significant lag phase and reached biomass 3. The biomass kept more or less the same at the same level up to 15 hours of the cultivation and slightly decreased thereafter reaching about 3. During exponential cell growth, the medium pH decreased gradually and reached its minimal value of about 4. After that time, the pH remained the same in the range of 4.

On the other hand, the alcohol accumulated at a very slow rate during the first 12 hours reaching about 0. The alcohol production rate increased gradually thereafter and reached its maximal value of 0. Based on these data, cultivation was conducted in L stirred tank bioreactor using this optimized medium to study the scalability and the industrial potential of this process. As shown in Figure 7, cells of S. The maximal cell mass 0f 8. However, the dissolved oxygen decreased rapidly during the first 12 hours and maintain in the range between