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).

Representative 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, 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.

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. In addition, they do not produce metabolites that can negatively affect the gastrointestinal tract. Besides, Choi and Chang²⁵, studied the L. plantarum EM and reported its potential for cholesterol reduction based on its bile salt hydrolase activity and cell surface binding ability. In addition to exhibiting tolerance to acid and bile stresses, L. plantarum EM also showed antimicrobial activity against pathogens and antibiotic resistance, validating its potential as a probiotic.

However, producing dried probiotics for commercialization is challenging since the microorganisms are exposed to various stress factors, such as thermal, mechanical, osmotic, and oxidative stresses. The high temperatures involved in the process can promote the denaturation of the enzymes and proteins involved in the cell's metabolism, causing microbial viability losses. Water removal during drying is also a critical factor since a minimum water content is necessary to sustain essential metabolic activity²⁶. The high shear forces caused by the passage of the probiotic mixture through the atomizer during the SD can also damage the probiotic cell's structure, contributing to viability losses^27,28. Hence, the correct selection of the SD operating conditions (e.g., the inlet and outlet drying temperatures, drying gas flow rate, feed flow rate of the probiotic composition, atomizing pressure, and gas flow rate) is essential to minimize the cell viability losses during spray drying, improve the product quality, and, thus, obtain acceptable dryer performance.

The composition of the constituents loaded with the probiotics is also a relevant factor since poorly designed formulations do not protect the probiotics during drying and storage, causing significant viability losses. The composition properties are improved by the addition of the so-called drying aids (or protectant agents), which can provide certain protection to the microorganism cells during the SD and storage^26,29. Carbohydrates (e.g., monosaccharides, disaccharides, polysaccharides, oligosaccharides, etc.), proteins, and reconstituted skim milk are usually added to the probiotic composition to protect the microorganism cells during SD. Although unclear, the protective effect of skim milk is associated with its complex composition, as it contains lactose, fat, casein, whey protein, and Ca²⁺cations; some authors have argued that the whey proteins and Ca²⁺ have a more prominent effect than the lactose^30,31. According to Fu et al.¹⁷, the use of skim milk with added whey confers high thermal protection to probiotics due to hydrophobic interactions between the dairy proteins and bacterial cells¹⁷.

The protective effects of the drying aids on probiotic viability are explained by three hypotheses used to justify the maintenance of protein conformation and enzyme activities during SD, namely the vitrification theory, the water replacement hypothesis, and the hydration forces hypothesis, which have been fully discussed by Broeckx et al.³⁰.

Stressing the probiotics during cultivation is another method that can be used to enhance the probiotic cell resistance during SD.

In the present protocol, the effects of protectant systems (a mixture of inulin:maltodextrin and simulated skim milk), and the spray-drying temperature on the viability and properties of the dried probiotic, as well as the SD performance, were evaluated.

The choice of simulated skim milk was based on the work of Písecký²². The fructooligosaccharide inulin and lactose were added as carbohydrates (instead of only lactose), and silicon dioxide was added as the ash. The choice of inulin was based on the literature, where it has been described as a prebiotic agent that can improve the benefits of the probiotics in the gut^32,33. The comparison of the protective effects of these drying aids on probiotics viability after SD was conducted at 80 °C. The results showed that the simulated skim milk promoted a higher probiotic viability than the inulin:maltodextrin combination at 80 °C. Hence, a study of the effects of the drying temperature (80 °C to 160 °C) on probiotic viability and powder yield was carried out with the simulated skim milk. As shown in Figure 1, the increase in the inlet temperature, which led to a higher outlet temperature, reduced the bacteria's survival, as expected¹⁷. However, the powder yield did not change at any temperature, remaining around 50%.

The dehydration level of probiotics is also linked to their viability losses during drying and product storage. The physical and chemical deteriorative reactions of a dried product depend on the free water level³⁴, but excessive dehydration can significantly reduce the viability of the dried probiotic. As expected, the increase in the drying temperature promoted a reduction in the product moisture content and water activity, which reached values of 3.01% ± 0.30% (w/w) and 0.201 ± 0.006 at 160 °C, respectively. Values of Aw below the monolayer moisture content (~0.40) are usually associated with a longer shelf-life due to the reduction in the free water available for biochemical reactions and microbial growth³⁴. However, at very low water activities (<0.20), lipid peroxidation reactions increase significantly, which can be detrimental to the product viability during storage. In terms of the moisture content, it is preferable that the values remain in the range of 2.8% to 5.6% to guarantee the preservation of the probiotics and reduce the deteriorative biochemical reactions in the long term^35,36.

Figure 2 shows that spray-drying temperatures above 120 °C are required to produce a product with the recommended Aw. At this temperature, the dried probiotic showed a viability of around 90%, a moisture content of 4.6 % w/w, and an Aw of 0.26, which are excellent results. Martins et al.³⁷, in an optimization study of the spray-drying of Lactococcus lactis cells, recommended an Aw value of 0.198 and an inlet spray drying temperature of 126 °C to minimize the microorganism viability losses, which are in close agreement with the values from this protocol.

Other powder characterization methodologies could be performed, such as to examine the morphological characteristics, stickiness³⁶, flowability, and compressibility³⁸.

In this context, spray drying temperatures above 120 °C are required to guarantee the viability and shelf life of microbial cells in a powdered preparation and their survival during food processing and storage. In industrial terms, this is an excellent result, as spray-drying technology is low-cost compared to freeze-drying, thus reducing the product price. Additionally, survival above 50% seems to be a robust range that guarantees probiotic powder functionality²⁸, meaning the survival is a good indicator for reproducing this protocol on an industrial scale. However, scaling up to industrial conditions needs to be tested to ensure the product has the same characteristics as the obtained powder in this protocol.

The methods described in this protocol aimed to clarify the importance of correct selection of the composition and processing the variables during the spray-drying of probiotic bacteria to ensure the viability and stability of the powder.

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