Hi,

When purifying exopolysaccharides (EPS) from bacterial extracts, deactivating enzymes is a critical step to ensure the integrity and bioactivity of the EPS. Heating the extraction solution to 95 °C is a common practice for enzyme deactivation, but this raises the question of whether this temperature will affect the bioactivity of the EPS.

Different EPS have varying thermal stabilities depending on their chemical structure. Generally, heating to 95 °C is unlikely to significantly impact the structure of polysaccharides because they often possess high thermal stability. Studies have shown that many bacterial EPS, such as those produced by **Lactobacillus** species, can withstand temperatures up to 100 °C for short periods without losing their bioactive properties (Freitas et al., 2011).

The purpose of thermal treatment at 95 °C is to denature enzymes and halt their activity, preventing further modification or degradation of the EPS during subsequent purification steps. This temperature is sufficient to denature most enzymes but usually not high enough to significantly affect the structure and bioactivity of polysaccharides.

However, the duration of heating is an important factor to consider. While brief heating (a few minutes) is generally not problematic, prolonged exposure to high temperatures could potentially degrade the EPS. Therefore, it is advisable to limit the heating time to the minimum necessary to effectively denature the enzymes without damaging the EPS.

In summary, heating to 95 °C for short periods is unlikely to significantly impact the bioactivity of the EPS. It is crucial to control the heating time to minimize potential negative effects. To ensure the EPS's bioactivity remains intact, specific bioactivity tests before and after thermal treatment can be conducted to detect and evaluate any changes.

References:

Freitas, F., Alves, V. D., & Reis, M. A. (2011). Advances in bacterial exopolysaccharides: from production to biotechnological applications. Trends in Biotechnology, 29(8), 388-398.

Phil Käding How much time do you think I should heat my EPS extract

furthermore, Can I use protease as an alternative solution?

To prepare a hyper-sweet medium for screening EPS (exopolysaccharide) producing lactic acid bacteria (LAB) using MRS broth, follow these steps:

Start by using De Man-Rogosa-Sharpe (MRS) broth as the base medium. For the carbohydrate source, you can use either sucrose or glucose, depending on your specific requirements and the LAB strain you are working with.

For sucrose, studies have shown that high concentrations are effective in promoting EPS production. You should aim for a concentration between 80 g/L and 136.5 g/L. To prepare the medium, dissolve the appropriate amount of sucrose into the MRS broth. For instance, adding 80 g/L to 136.5 g/L of sucrose to the MRS broth has been shown to enhance EPS yield effectively, as demonstrated in various studies (Valdez et al., 2020), (Seesuriyachan et al., 2012).

If you prefer using glucose, you should aim for a concentration around 20-40 g/L. For example, a concentration of 29.6 g/L of glucose has been found effective in optimizing conditions for EPS production (Abdi et al., 2012).

Once you have prepared the MRS broth with the desired concentration of sucrose or glucose, proceed to sterilize the medium by autoclaving it at 121°C for 15-20 minutes. After sterilization, allow the medium to cool to room temperature before use.

An example of a sucrose-based hyper-sweet MRS medium would involve preparing the MRS broth according to standard procedures and then adding 80 g/L to 136.5 g/L of sucrose. This medium is then autoclaved and cooled before inoculation.

Similarly, an example of a glucose-based hyper-sweet MRS medium involves preparing the MRS broth, adding 20 g/L to 40 g/L of glucose, autoclaving the medium, and cooling it down before use.

After preparing the medium, inoculate it with your LAB and incubate under optimal conditions, such as the appropriate temperature and pH for your specific strain. It may be necessary to optimize the sucrose or glucose concentration and other medium components based on the specific LAB strain being used.

By following these steps, you can create an effective hyper-sweet medium for screening EPS producing LAB, ensuring optimal conditions for maximum EPS production.

References:

• Valdez, A., Delgado, O., & Fariña, J. (2020). Cost-effective optimized scleroglucan production by Sclerotium rolfsii ATCC 201126 at bioreactor scale. International Journal of Biological Macromolecules.
• Seesuriyachan, P., Kuntiya, A., Hanmoungjai, P., Techapun, C., Chaiyaso, T., & Leksawasdi, N. (2012). Optimization of Exopolysaccharide Overproduction by Lactobacillus confusus in Solid State Fermentation under High Salinity Stress. Bioscience, Biotechnology, and Biochemistry.
• Abdi, R., Sheikh-Zeinoddin, M., & Soleimanian-Zad, S. (2012). Efficiency of modified skimmed milk base media to achieve high exopolysaccharide/cell ratios by Lactobacillus delbrueckii subsp. bulgaricus SZ2 in optimized conditions defined by the response surface methodology. International Journal of Food Science and Technology.

Phil Käding

I can't thank you enough

and for the type of sucre and extraction condition as you said I chose it depending on some results in some research

still how much inoculum do I need to add research has shown that the best inoculum size is 2 % but how milliliter equals this percentage I don't know

To deactivate enzymes in bacterial extract exopolysaccharide (EPS) without losing bioactivity, heating at 70-90°C for short durations is generally effective. Different enzymes have varying heat resistances, so specific experiments may be necessary to determine optimal conditions. Non-thermal alternatives can also be explored to preserve EPS quality while inactivating enzymes.

Abdelhak Maghchiche

barak allaho fika