Process Development for the Spray-Drying of Probiotic Bacteria and Evaluation of the Product Quality

Summary

This protocol details the steps involved in the production and physicochemical characterization of a spray-dried probiotic product.

Abstract

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.

Introduction

To be defined as probiotics, microorganisms added to foods (or supplements) have to be consumed alive, be able to survive during passage in the gastrointestinal tract of the host, and reach the site of action in adequate amounts to exert beneficial effects^1,2,7.

The growing interest in probiotics is due to the several benefits to human health they confer, such as the stimulation of the immune system, the reduction of serum cholesterol levels, and the enhancement of gut barrier function by acting against harmful microbes, as well as their beneficial effects in the treatment of the irritable bowel syndrome, among others^2,3. In addition, several studies have demonstrated that probiotics can positively affect other parts of the human body where unbalanced microbial communities can cause infectious diseases^3,4,5.

For probiotics to be therapeutically effective, the product should contain between 10⁶-10⁷ CFU/g of bacteria at the time of consumption⁶. On the other hand, the Italian Ministry of Health and Health Canada have established that the minimum level of probiotics in food should be 10⁹ CFU/g of viable cells per day or per serving, respectively⁷. Considering high loads of probiotics are needed to guarantee they will have beneficial effects, it is essential to guarantee their survival during processing, shelf storage, and passage through the gastrointestinal (GI) tract. Several studies have demonstrated that microencapsulation is an effective method to improve the overall viability of probiotics^8,9,10,11.

In this context, several methods have been developed for the microencapsulation of probiotics, such as spray-drying, freeze-drying, spray-chilling, emulsion, extrusion, coacervation, and, more recently, fluidized beds^11,12,13,14. Microencapsulation by spray-drying (SD) is widely used in the food industry because it is a simple, fast, and reproducible process. It is easy to scale up, and it has a high production yield at low energy requirements^11,12,13,14. Nonetheless, the exposure to high temperatures and low moisture content can affect the survival and viability of the probiotic cells¹⁵. Both parameters can be improved for a given strain by determining the effects of culture age and conditions to pre-adapt the culture and optimize the spray-drying conditions (inlet and outlet temperatures, atomization process) and the encapsulating composition^{8,14,16,17,18}.

The composition of the encapsulating solution is also an important factor during SD as it can define the level of protection against adverse environmental conditions. Inulin, Arabic gum, maltodextrins, and skim milk are widely used as encapsulating agents for probiotic drying^5,17,18,19. Inulin is a fructooligosaccharide that presents a strong prebiotic activity and promotes intestinal health¹⁹. Skim milk is very effective in maintaining the viability of dried bacterial cells and generates a powder with good reconstitution properties¹⁷.

Lactiplantibacillus paraplantarum FT-259 is a lactic acid bacterium that produces bacteriocin and presents antilisterial activity, besides probiotic traits^20,21. It is a facultative heterofermentative rod-shaped Gram-positive bacterium that grows from 15 °C to 37 °C²⁰ and is compatible with the homeostatic body temperature. This study aimed to present all the steps involved in the production and physicochemical characterization of a spray-dried probiotic (L. paraplantarum FT-259) and evaluate the influence of the protectants and drying temperatures.

Protocol

1. Production of the probiotic cells

1. Prepare De Man Rogosa and Sharpe (MRS) broth.
2. Reactivate 1% (v/v) of the culture of interest in the MRS broth (here, Lactiplantibacillus paraplantarum FT-259 was used).
3. Incubate for 24 h at an adequate temperature (we used 37 °C).

2. Separate the bacteria from the culture

1. 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.
2. 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.
3. Repeat the centrifugation process as mentioned before.
4. To obtain the pellet, use a pipette to remove and discard the supernatant in an appropriate container.

3. Addition of drying aids

1. Select the combination of two drying aid compositions (protectants): inulin:maltodextrin mixture and simulated skim milk (Table 1)^22,23.
2. Weigh 5 g of inulin and 5 g of maltodextrin to obtain the first combination of protectants.
3. 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.
4. Add each of the drying aids to ultrapure water (1:10), and submit to magnetic stirring until solubilization.
5. 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

1. 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
2. Prepare the protectants composition and add the concentrated probiotic pellets.
3. Start the feed of the probiotic composition (cells plus protectants) through a peristaltic pump.
4. Start the timer, and place the product-collecting vessel when the solution enters the atomizer.
5. Register the outlet temperature every 5 min to track possible temperature instabilities.
6. Stop the timer when all the probiotic composition has been fed to the SD.
7. 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.
8. 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

1. Product moisture content
   1. Precisely weigh 100 mg of the dried product, and place it in the titration vessel of the Karl-Fischer equipment.
   2. Press the initiation button to initiate the bi-amperometric titration of the water present in the sample.
2. Water activity
   1. Weigh 0.6 g of the dried product in the sample compartment of the hygrometer at 25 °C.
   2. 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

1. Dilute the previously prepared bacterial suspensions in 9 mL of peptone water (0.1%, v/v).
2. Vortex until complete dispersion.
3. Perform serial decimal dilutions (1:10) in 9 mL of saline solution (0.9% NaCl).
4. Seed the dilutions onto MRS agar plates, and incubate at 37 °C for 24-48 h.
5. Count the colony-forming units (CFU/g) using a colony counter with magnifying lens.
6. Calculate the probiotic viability in the dried product according to the following equation:
   EE (%) = (N∕N_o) × 100
   where, N is the number of viable cells after spray drying, and N_o is the number of bacterial cells before spray drying.
7. Express the number of viable cells in CFU/g of product dispersion.

7. Data analysis

1. Tabulate the obtained data in statistical software, and perform the analysis using a multiple comparison test (ANOVA).

Results

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 viability^17,19.

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, wi...

Discussion

L. paraplantarum FT-259 is a Gram-positive, rod-shaped bacterium, is a producer of bacteriocins with antilisterial activity, and has high probiotic potential²⁰. Son et al.²⁴ previously demonstrated the immunostimulant and antioxidant capacity of L. paraplantarum strains. Besides, they have great probiotic potential, with properties such as stability under artificial gastric and bile conditions, susceptibility to antibiotics, and binding to intestinal cells...

Materials

Name	Company	Catalog Number	Comments
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