Process Development for the Spray-Drying of Probiotic Bacteria and Evaluation of the Product Quality
PREPARAZIONE ISTRUTTORI
CONCETTI
Student Protocol
1. Production of the probiotic cells
- Prepare De Man Rogosa and Sharpe (MRS) broth.
- Reactivate 1% (v/v) of the culture of interest in the MRS broth (here, Lactiplantibacillus paraplantarum FT-259 was used).
- Incubate for 24 h at an adequate temperature (we used 37 °C).
2. Separate the bacteria from the culture
- Centrifuge the bacterial culture at 7,197 x g for 5 min at 4 °C using 50 mL conical tubes. It is important that the weight of the tubes is balanced before the procedure.
- Using a pipette, remove the supernatant, and discard it in a suitable container. Wash the pellets with a phosphate buffer (pH 7), and homogenize the solution.
- Repeat the centrifugation process as mentioned before.
- To obtain the pellet, use a pipette to remove and discard the supernatant in an appropriate container.
3. Addition of drying aids
- Select the combination of two drying aid compositions (protectants): inulin:maltodextrin mixture and simulated skim milk (Table 1)22,23.
- Weigh 5 g of inulin and 5 g of maltodextrin to obtain the first combination of protectants.
- Weigh 3 g of inulin, 3 g of lactose, 0.4 g of colloidal SiO2, and 3.6 g of whey protein to obtain the second combination of protectants.
- Add each of the drying aids to ultrapure water (1:10), and submit to magnetic stirring until solubilization.
- Ensure that the protectants and the water are homogeneous, then add the probiotics pellets to the mixture, and stir moderately for 20 min.
| Drying aids | Inulin and maltodextrin | Simulated skim milk |
| Maltodextrin | 5% | – |
| Whey protein | – | 3.60% |
| Lactose | – | 3% |
| Inulin | 5% | 3% |
| Colloidal SiO2 | – | 0.40% |
Table 1: Composition of the drying aids.
4. Spray-drying
- Turn on the spray dryer (SD), and set the drying gas flow rate, the inlet drying temperature, and the atomizer gas flow rate and pressure as follows:
Inlet temperature: 80 °C
Air flow: 60 m³/h
Feed rate: 4 g/min
Atomization flow: 17 L/min
Atomization pressure: 1.5 kgf/cm²
Diameter of the atomizer nozzle: 1 mm - Prepare the protectants composition and add the concentrated probiotic pellets.
- Start the feed of the probiotic composition (cells plus protectants) through a peristaltic pump.
- Start the timer, and place the product-collecting vessel when the solution enters the atomizer.
- Register the outlet temperature every 5 min to track possible temperature instabilities.
- Stop the timer when all the probiotic composition has been fed to the SD.
- Weigh the product-collecting vessel to determine the amount of composition fed to the system and the amount of dry product collected, to calculate the drying yield (product recovered) through a mass balance in the dryer.
- Use simulated skim milk to evaluate the effect of temperature on the viability of the probiotic cells, by setting up five different spray-drying temperatures (80 °C, 100 °C, 120 °C, 140 °C, and 160 °C vs. outlet temperatures of 59 °C, 70 °C, 83 °C, 96 °C, and 108 °C).
5. Powder characterization
- Product moisture content
- Precisely weigh 100 mg of the dried product, and place it in the titration vessel of the Karl-Fischer equipment.
- Press the initiation button to initiate the bi-amperometric titration of the water present in the sample.
- Water activity
- Weigh 0.6 g of the dried product in the sample compartment of the hygrometer at 25 °C.
- Close the equipment cover.
NOTE: The test will start automatically and stop when the sample reaches the equilibrium vapor pressure within the sample compartment.
6. Probiotic viability
- Dilute the previously prepared bacterial suspensions in 9 mL of peptone water (0.1%, v/v).
- Vortex until complete dispersion.
- Perform serial decimal dilutions (1:10) in 9 mL of saline solution (0.9% NaCl).
- Seed the dilutions onto MRS agar plates, and incubate at 37 °C for 24-48 h.
- Count the colony-forming units (CFU/g) using a colony counter with magnifying lens.
- Calculate the probiotic viability in the dried product according to the following equation:
EE (%) = (N∕No) × 100
where, N is the number of viable cells after spray drying, and No is the number of bacterial cells before spray drying. - Express the number of viable cells in CFU/g of product dispersion.
7. Data analysis
- Tabulate the obtained data in statistical software, and perform the analysis using a multiple comparison test (ANOVA).
Process Development for the Spray-Drying of Probiotic Bacteria and Evaluation of the Product Quality
Learning Objectives
In this study, L. paraplantarum was encapsulated by SD using food-grade encapsulating agents (inulin:maltodextrin and simulated milk powder), showing high product quality and efficacy in preserving the bacterial cell viability17,19.
The results of the SD of probiotics at 80 °C showed that the distinct protectants systems (inulin:maltodextrin and simulated skim milk) promoted efficient protection of the probiotic cells, with viabilities of 95.1% and 97.0%, respectively. The product yield was near 50% w/w for both protectants systems and was slightly superior for the simulated skim milk, which generated a product with a better appearance and flowability. Then, the probiotic composition combined with the simulated skim milk was submitted to spray-drying at higher temperatures from 80 °C to 160 °C (Figure 1).
As expected, the increase in SD temperature tended to decrease the probiotic viability, which reached nearly 80% at 160 °C. It can also be seen in Figure 1 that the effect of the drying temperature on the product yield was negligible, with an average value of 50.7% ± 2.4% w/w; these values are commonly observed for lab-scale spray dryers. These results indicate that the simulated skim milk is a good protectant system for probiotic drying, as it generates a high-quality product with good system performance (product yield).
The powders' moisture content and water activity decreased conversely with the spray-drying temperature, as expected (Figure 2).
Figure 1: Powder yield (%) and probiotic viability (%) according to the SD temperature (°C), with simulated skim milk as the drying aid. Please click here to view a larger version of this figure.
Figure 2: Moisture content and water activity of the dried probiotic samples according to the SD temperature (°C), with simulated skim milk as the protectant system. Please click here to view a larger version of this figure.
List of Materials
| Aqua Lab 4TEV | Decagon Devices | – | Water activity meter |
| Centrifuge (mod. 5430 R ) | Eppendorf | – | Centrifuge |
| Colloidal SiO2 (Aerosil 200) | Evokik | 7631-86-9 | drying aid |
| Fructooligosaccharides from chicory | Sigma-Aldrich | 9005-80-5 | drying aid |
| GraphPad Prism (version 8.0) software | GraphPad Software | – | San Diego, California, USA |
| Karl Fischer 870 Titrino Plus | Metrohm | – | Moisture content |
| Lactose | Milkaut | 63-42-3 | drying aid |
| Maltodextrin | Ingredion | 9050-36-6 | drying aid |
| Milli-Q | Merk | – | Ultrapure water system |
| MRS Agar | Oxoid | – | Culture medium |
| MRS Broth | Oxoid | – | Culture medium |
| OriginPro (version 9.0) software | OriginLab | – | Northampton, Massachusetts, USA |
| Spray dryer SD-05 | Lab-Plant Ltd | – | Spray dryer |
| Whey protein | Arla Foods Ingredients S.A. | 91082-88-1 | drying aid |
Lab Prep
Probiotics and prebiotics are of great interest to the food and pharmaceutical industries due to their health benefits. Probiotics are live bacteria that can confer beneficial effects on human and animal wellbeing, while prebiotics are types of nutrients that feed the beneficial gut bacteria. Powder probiotics have gained popularity due to the ease and practicality of their ingestion and incorporation into the diet as a food supplement. However, the drying process interferes with cell viability since high temperatures inactivate probiotic bacteria. In this context, this study aimed to present all the steps involved in the production and physicochemical characterization of a spray-dried probiotic and evaluate the influence of the protectants (simulated skim milk and inulin:maltodextrin association) and drying temperatures in increasing the powder yield and cell viability. The results showed that the simulated skim milk promoted higher probiotic viability at 80 °C. With this protectant, the probiotic viability, moisture content, and water activity (Aw) reduce as long as the inlet temperature increases. The probiotics' viability decreases conversely with the drying temperature. At temperatures close to 120 °C, the dried probiotic showed viability around 90%, a moisture content of 4.6% w/w, and an Aw of 0.26; values adequate to guarantee product stability. In this context, spray-drying temperatures above 120 °C are required to ensure the microbial cells' viability and shelf-life in the powdered preparation and survival during food processing and storage.
Probiotics and prebiotics are of great interest to the food and pharmaceutical industries due to their health benefits. Probiotics are live bacteria that can confer beneficial effects on human and animal wellbeing, while prebiotics are types of nutrients that feed the beneficial gut bacteria. Powder probiotics have gained popularity due to the ease and practicality of their ingestion and incorporation into the diet as a food supplement. However, the drying process interferes with cell viability since high temperatures inactivate probiotic bacteria. In this context, this study aimed to present all the steps involved in the production and physicochemical characterization of a spray-dried probiotic and evaluate the influence of the protectants (simulated skim milk and inulin:maltodextrin association) and drying temperatures in increasing the powder yield and cell viability. The results showed that the simulated skim milk promoted higher probiotic viability at 80 °C. With this protectant, the probiotic viability, moisture content, and water activity (Aw) reduce as long as the inlet temperature increases. The probiotics' viability decreases conversely with the drying temperature. At temperatures close to 120 °C, the dried probiotic showed viability around 90%, a moisture content of 4.6% w/w, and an Aw of 0.26; values adequate to guarantee product stability. In this context, spray-drying temperatures above 120 °C are required to ensure the microbial cells' viability and shelf-life in the powdered preparation and survival during food processing and storage.
Procedura
Probiotics and prebiotics are of great interest to the food and pharmaceutical industries due to their health benefits. Probiotics are live bacteria that can confer beneficial effects on human and animal wellbeing, while prebiotics are types of nutrients that feed the beneficial gut bacteria. Powder probiotics have gained popularity due to the ease and practicality of their ingestion and incorporation into the diet as a food supplement. However, the drying process interferes with cell viability since high temperatures inactivate probiotic bacteria. In this context, this study aimed to present all the steps involved in the production and physicochemical characterization of a spray-dried probiotic and evaluate the influence of the protectants (simulated skim milk and inulin:maltodextrin association) and drying temperatures in increasing the powder yield and cell viability. The results showed that the simulated skim milk promoted higher probiotic viability at 80 °C. With this protectant, the probiotic viability, moisture content, and water activity (Aw) reduce as long as the inlet temperature increases. The probiotics' viability decreases conversely with the drying temperature. At temperatures close to 120 °C, the dried probiotic showed viability around 90%, a moisture content of 4.6% w/w, and an Aw of 0.26; values adequate to guarantee product stability. In this context, spray-drying temperatures above 120 °C are required to ensure the microbial cells' viability and shelf-life in the powdered preparation and survival during food processing and storage.