This study was financed in part by the Coordena&#231;&#227;o de Aperfei&#231;oamento de Pessoal de N&#237;vel Superior - Brasil (CAPES) - Finance Code 001. This study was also supported in part by FAPESP - S&#227;o Paulo Research Foundation. E.C.P.D.M. is grateful for a Researcher Fellowship from National Council for Scientific and Technological Development (CNPq) 306330/2019-9.

1. Production of the probiotic cells
<ol>
	<li>Prepare De Man Rogosa and Sharpe (MRS) broth.</li>
	<li>Reactivate 1% (v/v) of the culture of interest in the MRS broth (here, Lactiplantibacillus paraplantarum FT-259 was used).</li>
	<li>Incubate for 24 h at an adequate temperature (we used 37 &#176;C).</li>
</ol>
2. Separate the bacteria from the culture
<ol>
	<li>Centrifuge the bacterial culture at 7,197 x g for 5 min at 4 &#176;C using 50 mL conical tubes. It is important that the weight of the tubes is balanced before the procedure.</li>
	<li>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.</li>
	<li>Repeat the centrifugation process as mentioned before.</li>
	<li>To obtain the pellet, use a pipette to remove and discard the supernatant in an appropriate container.</li>
</ol>
3. Addition of drying aids
<ol>
	<li>Select the combination of two drying aid compositions (protectants): inulin:maltodextrin mixture and simulated skim milk (Table 1)22,23.</li>
	<li>Weigh 5 g of inulin and 5 g of maltodextrin to obtain the first combination of protectants.</li>
	<li>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.</li>
	<li>Add each of the drying aids to ultrapure water (1:10), and submit to magnetic stirring until solubilization.</li>
	<li>Ensure that the protectants and the water are homogeneous, then add the probiotics pellets to the mixture, and stir moderately for 20 min.</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Drying aids</td>
			<td>Inulin and maltodextrin</td>
			<td>Simulated skim milk</td>
		</tr>
		<tr>
			<td>Maltodextrin</td>
			<td>5%</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Whey protein</td>
			<td>-</td>
			<td>3.60%</td>
		</tr>
		<tr>
			<td>Lactose</td>
			<td>-</td>
			<td>3%</td>
		</tr>
		<tr>
			<td>Inulin</td>
			<td>5%</td>
			<td>3%</td>
		</tr>
		<tr>
			<td>Colloidal SiO2</td>
			<td>-</td>
			<td>0.40%</td>
		</tr>
	</tbody>
</table>
Table 1: Composition of the drying aids.
4. Spray-drying
<ol>
	<li>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 &#176;C 
	Air flow: 60 m&#179;/h 
	Feed rate: 4 g/min 
	Atomization flow: 17 L/min 
	Atomization pressure: 1.5 kgf/cm&#178; 
	Diameter of the atomizer nozzle: 1 mm</li>
	<li>Prepare the protectants composition and add the concentrated probiotic pellets.</li>
	<li>Start the feed of the probiotic composition (cells plus protectants) through a peristaltic pump.</li>
	<li>Start the timer, and place the product-collecting vessel when the solution enters the atomizer.</li>
	<li>Register the outlet temperature every 5 min to track possible temperature instabilities.</li>
	<li>Stop the timer when all the probiotic composition has been fed to the SD.</li>
	<li>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.</li>
	<li>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 &#176;C, 100 &#176;C, 120 &#176;C, 140 &#176;C, and 160 &#176;C vs.&#160;outlet temperatures of 59 &#176;C, 70 &#176;C, 83 &#176;C, 96 &#176;C, and 108 &#176;C).</li>
</ol>
5. Powder characterization
<ol>
	<li>Product moisture content
	<ol>
		<li>Precisely weigh 100 mg of the dried product, and place it in the titration vessel of the Karl-Fischer equipment.</li>
		<li>Press the initiation button to initiate the bi-amperometric titration of the water present in the sample.</li>
	</ol>
	</li>
	<li>Water activity
	<ol>
		<li>Weigh 0.6 g of the dried product in the sample compartment of the hygrometer at 25 &#176;C.</li>
		<li>Close the equipment cover. 
		&#8203;NOTE: The test will start automatically and stop when the sample reaches the equilibrium vapor pressure within the sample compartment.</li>
	</ol>
	</li>
</ol>
6. Probiotic viability
<ol>
	<li>Dilute the previously prepared bacterial suspensions in 9 mL of peptone water (0.1%, v/v).</li>
	<li>Vortex until complete dispersion.</li>
	<li>Perform serial decimal dilutions (1:10) in 9 mL of saline solution (0.9% NaCl).</li>
	<li>Seed the dilutions onto MRS agar plates, and incubate at 37 &#176;C for 24-48 h.</li>
	<li>Count the colony-forming units (CFU/g) using a colony counter with magnifying lens.</li>
	<li>Calculate the probiotic viability in the dried product according to the following equation: 
	EE (%) = (N&#8725;No) &#215; 100 
	where, N is the number of viable cells after spray drying, and No is the number of bacterial cells before spray drying.</li>
	<li>Express the number of viable cells in CFU/g of product dispersion.</li>
</ol>
7. Data analysis
<ol>
	<li>Tabulate the obtained data in statistical software, and perform the analysis using a multiple comparison test (ANOVA).</li>
</ol>

Optimizing Spray-Dried Probiotic Product Quality Through Process Development

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The authors have no conflicts of interest to declare.

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...

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 effects1,2,7.
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 others2,3. In addition, several studies have demonstrated that probiotics can positively affect other parts of the human body where unbalanced microbial communities can cause infectious diseases3,4,5.
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 probiotics8,9,10,11.
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 beds11,12,13,14. 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 requirements11,12,13,14. 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 composition8,14,16,17,18.
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 drying5,17,18,19. 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 traits20,21. It is a facultative heterofermentative rod-shaped Gram-positive bacterium that grows from 15 &#176;C to 37 &#176;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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Aqua Lab 4TEV</td>
			<td>Decagon Devices</td>
			<td>-</td>
			<td>Water activity meter</td>
		</tr>
		<tr>
			<td>Centrifuge (mod. 5430 R )</td>
			<td>Eppendorf</td>
			<td>-</td>
			<td>Centrifuge</td>
		</tr>
		<tr>
			<td>Colloidal SiO2 (Aerosil 200)</td>
			<td>Evokik</td>
			<td>7631-86-9</td>
			<td>drying aid</td>
		</tr>
		<tr>
			<td>Fructooligosaccharides from chicory</td>
			<td>Sigma-Aldrich</td>
			<td>9005-80-5</td>
			<td>drying aid</td>
		</tr>
		<tr>
			<td>GraphPad Prism (version 8.0) software</td>
			<td>GraphPad Software</td>
			<td>-</td>
			<td>San Diego, California, USA</td>
		</tr>
		<tr>
			<td>Karl Fischer 870 Titrino Plus</td>
			<td>Metrohm</td>
			<td>-</td>
			<td>Moisture content</td>
		</tr>
		<tr>
			<td>Lactose</td>
			<td>Milkaut</td>
			<td>63-42-3&#160;</td>
			<td>drying aid</td>
		</tr>
		<tr>
			<td>Maltodextrin</td>
			<td>Ingredion</td>
			<td>9050-36-6</td>
			<td>drying aid</td>
		</tr>
		<tr>
			<td>Milli-Q</td>
			<td>Merk</td>
			<td>-</td>
			<td>Ultrapure water system</td>
		</tr>
		<tr>
			<td>MRS Agar</td>
			<td>Oxoid</td>
			<td>-</td>
			<td>Culture medium</td>
		</tr>
		<tr>
			<td>MRS Broth</td>
			<td>Oxoid</td>
			<td>-</td>
			<td>Culture medium</td>
		</tr>
		<tr>
			<td>OriginPro (version 9.0) software</td>
			<td>OriginLab</td>
			<td>-</td>
			<td>Northampton, Massachusetts, USA</td>
		</tr>
		<tr>
			<td>Spray dryer SD-05</td>
			<td>Lab-Plant Ltd</td>
			<td>-</td>
			<td>Spray dryer</td>
		</tr>
		<tr>
			<td>Whey protein</td>
			<td>Arla Foods Ingredients S.A.</td>
			<td>91082-88-1</td>
			<td>drying aid</td>
		</tr>
	</tbody>
</table>

process development for the spray-drying of probiotic bacteria and evaluation of the product quality

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 &#176;C. With this protectant, the probiotic viability, moisture content, and water activity (Aw) reduce as long as the inlet temperature increases. The probiotics&#39; viability decreases conversely with the drying temperature.&#160;At temperatures close to 120 &#176;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 &#176;C are required to ensure the microbial cells&#39; viability and shelf-life in the powdered preparation and survival during food processing and storage.

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

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

In our laboratory, we do research on beneficial microbes, including probiotics, which are live microorganisms that can be added to food to improve the health of consumers, Usually by competing with pathogenic bacteria in the gut to exclude them and relieve symptoms, like diarrhea or other gastrointestinal disorders. Nowadays, many techniques have been applied to guarantee that the probiotics will survive in food during the shelf life. And also, they needed to tolerate the harsh conditions of the gastrointestinal tract to exert their beneficial effects inside the gut.We studied many technologies that can enhance the survival of probiotics, either in food or in the human host. Among the techniques, microencapsulation by spray drying is of great interest because it's simple, fast, and reproducible, providing high yields at low energy requirements. It's challenging, however, to determine the right conditions to obtain dried probiotics.And for that, suitable protectants must be chosen. Skim milk, maltodextrin, or other like probiotic inulin have been used with different probiotic strains. The area of microencapsulation is a happily evolving field of research that has the potential to impact the food industry by providing new products to be used as functional ingredients.In this study, we demonstrate how to obtain a microencapsulated preparation of a potential probiotic culture using the spray drying technique. We also show how to determine the main parameters to characterize the powder, obtain it, and how to evaluate the survival rate of the probiotic.

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 &#176;C showed that the distinct protectants systems (inulin:maltodextrin and simulated skim milk) promoted efficient protection of the probiotic cells, wi...

Watch this Scientific Journal Video about Process Development for the Spray-Drying of Probiotic Bacteria and Evaluation of the Product Quality at JoVE.com

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

Probiotics and prebiotics are of great interest to the food and pharmaceutical industries due to their health benefits. Probiotics are live bacteria that ...

process-development-for-spray-drying-probiotic-bacteria-evaluation

Research

JoVE Journal

Biology

﻿Addition of Drying Aids and Spray-Drying for Probiotic Assessment

Addition of Drying Aids and Spray-Drying for Probiotic Assessment  

To prepare the inulin-maltodextrin protectant combination, weigh five grams each of inulin and maltodextrin. To prepare the second combination of protectants, weigh three grams of inulin, three grams of lactose, 0.4 grams of colloidal silicon dioxide and 3.6 grams of whey protein. After weighing, add each of the drying aids to ultrapure water and stir on a magnetic stir until solubilization.Once the solution is homogenous, add probiotic pellets prepared from lactiplantibacillus paraplantarum FT259 bacterial culture to the mixture and stir moderately for 20 minutes. For spray drying, turn on the spray dryer. Set the inlet temperature to 120 degrees Celsius.Then, set the outlet temperature to 83 degrees Celsius. Set the airflow to 60 cubic meters per hour. Feed rate to four grams per minute.Atomization flow to 17 liters per minute. Atomization pressure to 1.5 kilogram force per square centimeter. And diameter of the atomizer nozzle to one millimeter.Once all the parameters are set, place the two combinations of protectants in the spray dryer. Start the feed of probiotic composition through a peristaltic pump. Then, start the timer and place the product collecting vessel when the solution enters the atomizer.Record the outlet temperature every five minutes to track possible temperature instabilities. Stop the timer when all the probiotic composition has been fed to the spray dryer. Weigh the product collecting vessel to determine the amount of composition fed to the system and the amount of dry product collected.

Powder Characterization and Probiotic Viability

To determine product moisture content, precisely weigh 100 milligrams of the dried product and place it in the titration vessel of Karl Fisher Equipment. Initiate the biamperometric titration of water present in the sample by pressing the initiation button. To determine water activity, weigh 0.6 grams of dried product in the sample compartment of the hygrometer at 25 degrees Celsius and close the equipment cover.For determining probiotic viability, dilute bacterial culture in nine milliliters of peptide water and vortex until complete dispersion. Perform serial decimal dilutions with diluted bacterial culture in nine milliliters of saline solution. Seed the dilutions onto De Man, Rogosa and Sharpe agar plates and incubate at 37 degrees Celsius for 24 to 48 hours.After incubation, count the colony forming units or CFU using a colony counter with magnifying lens. Calculate the probiotic viability in the dried product using the given equation to express the number of viable cells in CFU per gram of product dispersion. The results of the spray drying of the probiotics at 80 degree Celsius showed that the distinct drying aids promoted efficient protection of the probiotic cells.The increase in spray drying temperature tended to decrease the probiotic viability and reached nearly 80%at 160 degrees Celsius. However, the powder yield remained around 50%throughout all the temperatures. The powder's moisture content and water activity decreased conversely with the spray drying temperature.

2.4K Views.  Universidade de São Paulo.  This protocol details the steps involved in the production and physicochemical characterization of a spray-dried probiotic product.

Optimizing Spray-Dried Probiotic Product Quality Through Process Development

Summary