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. 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&#39;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&#39;s structure, contributing to viability losses27,28. 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 storage26,29. 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 lactose30,31. 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 P&#237;seck&#253;22. 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 gut32,33. The comparison of the protective effects of these drying aids on probiotics viability after SD was conducted at 80 &#176;C. The results showed that the simulated skim milk promoted a higher probiotic viability than the inulin:maltodextrin combination at 80 &#176;C. Hence, a study of the effects of the drying temperature (80 &#176;C to 160 &#176;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&#39;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% &#177; 0.30% (w/w) and 0.201 &#177; 0.006 at 160 &#176;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 (&#60;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 term35,36.
Figure 2 shows that spray-drying temperatures above 120 &#176;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 &#176;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 &#176;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.

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><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 SiO&lt;sub&gt;2&lt;/sub&gt; (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&amp;nbsp;</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.

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, 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 &#176;C to 160 &#176;C (Figure 1).
As expected, the increase in SD temperature tended to decrease the probiotic viability, which reached nearly 80% at 160 &#176;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% &#177; 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&#160;for probiotic drying, as it generates a high-quality product with good system performance (product yield).
The powders&#39; moisture content and water activity decreased conversely with the spray-drying temperature, as expected (Figure 2).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65192/65192fig01v2.jpg" /> 
Figure 1: Powder yield (%) and probiotic viability (%) according to the SD temperature (&#176;C), with simulated skim milk as the drying aid. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65192/65192fig01largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65192/65192fig02.jpg" /> 
Figure 2: Moisture content and water activity of the dried probiotic samples according to the SD temperature (&#176;C), with simulated skim milk as the protectant system. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65192/65192fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

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

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

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Biology

3.2K Views.  Universidade de São Paulo. In ons laboratorium doen we onderzoek naar nuttige microben, waaronder probiotica, die levende micro-organismen zijn die aan voedsel kunnen worden toegevoegd om de gezondheid van consumenten te verbeteren, meestal door te concurreren met pathogene bacteriën in de darm om ze uit te sluiten en symptomen te verlichten, zoals diarree of andere gastro-intestinale stoornissen. Tegenwoordig zijn er veel technieken toegepast om te garanderen dat de probiotica tijdens de houdbaarheid in voedsel zulle...

Video: Auteur Spotlight: Procesontwikkeling voor het sproeidrogen van probiotische bacteriën en evaluatie van de productkwaliteit  

Measuring Volatile and Non-volatile Antifungal Activity of Biocontrol Products

The protocol described is based on a plug-transfer technique that allows accurate determination of microorganism quantities and their developmental stages. A specified number of spores are spread on an agar plate. This agar plate is incubated for a defined period to allow the fungi to reach the expected developmental stage, except for spores where incubation is not required. Agar plugs covered by spores, hyphae, or mycelium are next withdrawn and transferred onto agar media containing the antifungal compound to be tested either placed at a distance from the fungi or in contact. This method is applicable to test both liquid extracts and solid samples (powders). It is particularly well suited for quantifying the relative contributions of volatile and non-volatile agents in bioactive mixtures and for determining their effects, specifically on spores, early hyphae, and mycelium.
The method is highly relevant for the characterization of the antifungal activity of biocontrol products, notably plant-derived products. Indeed, for plant treatment, the results can be used to guide the choice of mode of application and to establish the trigger thresholds.

We describe a modified agar-based method designed to quantify the antifungal effects of plant-derived products. Both volatile and non-volatile contributions to the antifungal activity can be assessed through this protocol. In addition, efficacy against fungi can be measured at key developmental stages in a single experimental setup.

The protocol described is based on a plug-transfer technique that allows accurate determination of microorganism quantities and their developmental ...

Environment

Preparation of High-Quality Fermented Fish Product

This protocol provides a method for preparation of industrialized fermented fish product with sturgeon (Aquilaria sinensis) meat product. The procedures were: (1) pretreatment of farmed sturgeon including decapitation, evisceration, skinning-off, cleaning and cutting; (2) marinating fish cubes in 6-12% (w/v) salt solution (1:1, fish cube mass to solution volume); (3) drying fish cubes to a water content of 50-60% by hot air (40-60 &#176;C) or by vacuum; (4) fermentation involving inoculating fish cubes with 0.4-1.6% (w/w) S. cerevisiae in flavor solution to fish cubes and fermenting at 25-35 &#176;C for 6-10 h; (5) sealing fish cubes in vacuum packages with marinating and fermenting solutions; (6) sterilizing at 115-121 &#176;C for 10-20 min. The sturgeon meat product prepared by this method has delicious taste which is mellow and thick, has various types and large amounts of volatile flavor compounds such as alcohols and esters which could mask musty and unpleasant odor from fish, has moderate salt content but good texture properties such as high springiness, gumminess and chewiness, and has bright russet color and attractive appearance. This new technique could also be applied in the processing of other fish to provide convenient fish snack foods which could be stored at room temperature. It is appropriate for both marine and freshwater fish.

The goal of the protocol is to provide an industrialized fish fermentation technique based on inoculation of Saccharomyces cerevisiae.

This protocol provides a method for preparation of industrialized fermented fish product with sturgeon (Aquilaria sinensis) meat product. The procedures ...

Biochemistry

Visualizing the Beating Heart in Drosophila

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in myogenic hearts. A key step in examining heart function in the fly is finding a way to access the heart in a manner that preserves its myogenic function while still allowing the beating heart organ to be observed and recorded. Two different methods for observing and recording the beating heart in both larva and adult Drosophila are described here. Our semi-intact preparation using adult flies allows clear visualization of the abdominal heart without interference from the pigmented cuticle and overlying fat bodies. To record larval heart beats it is necessary to immobilize the larva, which minimizes body wall movements thereby reducing heart movements that are not associated with myocardial contractions. Our methodologies produce stable adult and larval heart preparations that can beat for hours at rates of 1-3 Hz.

Visualizing the Beating Heart in Drosophila

Technique required for visualizing the beating heart in larval and adult Drosophila are presented. Each life stage requires a different methodology.

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in ...

Generic Protocol for Optimization of Heterologous Protein Production Using Automated Microbioreactor Technology

A core business in industrial biotechnology using microbial production cell factories is the iterative process of strain engineering and optimization of bioprocess conditions. One important aspect is the improvement of cultivation medium to provide an optimal environment for microbial formation of the product of interest. It is well accepted that the media composition can dramatically influence overall bioprocess performance. Nutrition medium optimization is known to improve recombinant protein production with microbial systems and thus, this is a rewarding step in bioprocess development. However, very often standard media recipes are taken from literature, since tailor-made design of the cultivation medium is a tedious task that demands microbioreactor technology for sufficient cultivation throughput, fast product analytics, as well as support by lab robotics to enable reliability in liquid handling steps. Furthermore, advanced mathematical methods are required for rationally analyzing measurement data and efficiently designing parallel experiments such as to achieve optimal information content.
The generic nature of the presented protocol allows for easy adaption to different lab equipment, other expression hosts, and target proteins of interest, as well as further bioprocess parameters. Moreover, other optimization objectives like protein production rate, specific yield, or product quality can be chosen to fit the scope of other optimization studies. The applied Kriging Toolbox (KriKit) is a general tool for Design of Experiments (DOE) that contributes to improved holistic bioprocess optimization. It also supports multi-objective optimization which can be important in optimizing both upstream and downstream processes.

This manuscript describes a generic approach for tailor-made design of microbial cultivation media. This is enabled by an iterative workflow combining Kriging-based experimental design and microbioreactor technology for sufficient cultivation throughput, which is supported by lab robotics to increase reliability and speed in liquid handling media preparation.

A core business in industrial biotechnology using microbial production cell factories is the iterative process of strain engineering and optimization of ...

Bioengineering

Prospecting Microbial Strains for Bioremediation and Probiotics Development for Metaorganism Research and Preservation

Pollution affects all biomes. Marine environments have been particularly impacted, especially coral reefs, one of the most sensitive ecosystems on Earth. Globally, 4.5 billion people are economically dependent on the sea, where most of their livelihood is provided by coral reefs. Corals are of great importance and therefore their extinction leads to catastrophic consequences. There are several possible solutions to remediate marine pollutants and local contamination, including bioremediation. Bioremediation is the capacity of organisms to degrade contaminants. The approach presents several advantages, such as sustainability, relatively low cost, and the fact that it can be applied in different ecosystems, causing minimal impacts to the environment. As an extra advantage, the manipulation of endogenous microbiomes, including putative beneficial microorganisms for corals (pBMCs), may have probiotic effects for marine animals. In this context, the use of the two approaches, bioremediation and pBMC inoculation combined, could be promising. This strategy would promote the degradation of specific pollutants that can be harmful to corals and other metaorganisms while also increasing host resistance and resilience to deal with pollution and other threats. This method focuses on the selection of pBMCs to degrade two contaminants: the synthetic estrogen 17a-ethinylestradiol (EE2) and crude oil. Both have been reported to negatively impact marine animals, including corals, and humans. The protocol describes how to isolate and test bacteria capable of degrading the specific contaminants, followed by a description of how to detect some putative beneficial characteristics of these associated microbes to their coral host. The methodologies described here are relatively cheap, easy to perform, and highly adaptable. Almost any kind of soluble target compound can be used instead of EE2 and oil.

Pollution affects all biomes. Marine environments have been particularly impacted, especially coral reefs, one of the most sensitive ecosystems on Earth. Bioremediation is the capacity of organisms to degrade contaminants. Here, we describe methodologies to isolate and test microbes presenting bioremediation ability and potential probiotic characteristics for corals.

Pollution affects all biomes. Marine environments have been particularly impacted, especially coral reefs, one of the most sensitive ecosystems on Earth. ...

Formulations for Freeze-drying of Bacteria and Their Influence on Cell Survival

Cellular water can be removed to reversibly inactivate microorganisms to facilitate storage. One such method of removal is freeze-drying, which is considered a gentle dehydration method. To facilitate cell survival during drying, the cells are often formulated beforehand. The formulation forms a matrix that embeds the cells and protects them from various harmful stresses imposed on the cells during freezing and drying. We present here a general method to evaluate the survival rate of cells after freeze-drying and we illustrate it by comparing the results obtained with four different formulations: the disaccharide sucrose, the sucrose derived polymer Ficoll PM400, and the respective polysaccharides hydroxyethyl cellulose (HEC) and hydroxypropyl methyl cellulose (HPMC), on two strains of bacteria, P. putida KT2440 and A. chlorophenolicus A6. In this work we illustrate how to prepare formulations for freeze-drying and how to investigate the mechanisms of cell survival after rehydration by characterizing the formulation using of differential scanning calorimetry (DSC), surface tension measurements, X-ray analysis, and electron microscopy and relating those data to survival rates. The polymers were chosen to get a monomeric structure of the respective polysaccharide resembling sucrose to a varying degrees. Using this method setup we showed that polymers can support cell survival as effectively as disaccharides if certain physical properties of the formulation are controlled1.

Freeze-drying is often an easy and convenient way to obtain dry products of viable bacterial cells. An issue of the process is cell survival. We detail here a procedure to investigate how cell survival during freeze-drying is influenced by the properties of the formulation used.

Cellular water can be removed to reversibly inactivate microorganisms to facilitate storage. One such method of removal is freeze-drying, which is ...

Novel Production Protocol for Small-scale Manufacture of Probiotic Fermented Foods

A novel dried bacterial consortium of Lactobacillus rhamnosus yoba 2012 and Streptococcus thermophilus C106 is cultured in 1 L of milk. This fresh starter can be used for the production of fermented milk and other fermented foods either at home or at small-scale in rural settings. For the fresh starter, 1 L of milk is pasteurized in a pan that fits into a larger pan containing water, placed on a source of heat. In this water bath, the milk is heated and incubated at 85 &#176;C for 30 min. Thereafter, the milk is cooled down to 45 &#176;C, transferred to a vacuum flask, inoculated with the dried bacteria and left for at least 16 hr between 30 &#176;C and 45 &#176;C. For the purpose of frequent home production, the fresh starter is frozen into ice cubes, which can be used for the production of small volumes of up to 2 L of fermented milk. For the purpose of small-scale production in resource-poor countries, pasteurization of up to 100 L of milk is conducted in milk cans that are placed in a large sauce pan filled with water and heated on a fire at 85 &#176;C for 30 min, and subsequently cooled to 45 &#176;C. Next, the 100 L batch is inoculated with the 1 L freshly prepared starter mentioned before. To assure an effective fermentation at a temperature between 30 and 45 &#176;C, the milk can is covered with a blanket for 12 hr. For the production of non-dairy fermented foods, the fresh starter is left in a cheese cloth for 12 hr, and the drained-off whey can be subsequently used for the inoculation of a wide range of food raw materials, including vegetables and cereal-based foods.

A production protocol is described for the small-scale production of probiotic fermented dairy drink with the aid of a novel bacterial starter culture.

A novel dried bacterial consortium of Lactobacillus rhamnosus yoba 2012 and Streptococcus thermophilus C106 is cultured in 1 L of milk. This fresh starter ...

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure plasma (LPP) discharges contain a broad spectrum of active species, which lead to rapid microbial inactivation. To study the efficiency and mechanisms of sterilization by LPP, we use spores of the test organism Bacillus subtilis because of their extraordinary resistance against conventional sterilization procedures. We describe the production of B. subtilis spore monolayers, the sterilization process by low pressure plasma in a double inductively coupled plasma reactor, the characterization of spore morphology using scanning electron microscopy (SEM), and the analysis of germination and outgrowth of spores by live cell microscopy. A major target of plasma species is genomic material (DNA) and repair of plasma-induced DNA lesions upon spore revival is crucial for survival of the organism. Here, we study the germination capacity of spores and the role of DNA repair during spore germination and outgrowth after treatment with LPP by tracking fluorescently-labelled DNA repair proteins (RecA) with time-resolved confocal fluorescence microscopy. Treated and untreated spore monolayers are activated for germination and visualized with an inverted confocal live cell microscope over time to follow the reaction of individual spores. Our observations reveal that the fraction of germinating and outgrowing spores is dependent on the duration of LPP-treatment reaching a minimum after 120 s. RecA-YFP (yellow fluorescence protein) fluorescence was detected only in few spores and developed in all outgrowing cells with a slight elevation in LPP-treated spores. Moreover, some of the vegetative bacteria derived from LPP-treated spores showed an increase in cytoplasm and tended to lyse. The described methods for analysis of individual spores could be exemplary for the study of other aspects of spore germination and outgrowth.

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

This protocol illustrates the important consecutive steps required to assess the relevance of monitoring vitality parameter and DNA repair processes in reviving Bacillus subtilis spores after treatment with low pressure plasma by tracking fluorescence-labelled DNA repair proteins via time-resolved confocal microscopy and scanning electron microscopy.

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure ...

The Cultivation, Growth, and Viability of Lactic Acid Bacteria: A Quality Control Perspective

Lactic acid bacteria (LAB) are essential dairy starter cultures that are significantly employed for the manufacture of fermented dairy products such as yogurt and cheese. LAB predominantly produce lactic acid as a major end product of fermentation, and they synthesize important metabolites that impart the organoleptic characteristics of fermented food products. LAB are fastidious bacteria that thrive in many environments when adequate nutritional requirements are fulfilled. The demand for superior LAB dairy starter cultures for fermentation applications in the food and dairy industry, has resulted in the need to provide viable and active cultures for all bioprocessing operations. The development of a standard protocol for ensuring the viability and enhanced functionality of LAB cultures in the laboratory as well as dairy processing environments is thus very critical. In addressing concerns linked to resuscitating weak, stressed, and injured LAB culture cells, a protocol that vividly outlines salient steps to recover, enhance cell regeneration, and improve metabolic functionality of LAB strains is of the utmost importance. The maintenance of culture purity, functionality, and viability for LAB starter cultures is likewise critical. Therefore, adherence to a unique protocol guideline will result in the promotion of fermentation performance for many LAB strains dedicated to fermentation and biotechnology processes. As a result, the Food Microbiology and Biotechnology Laboratory at North Carolina Agriculture and Technical State University has developed a standard protocol for the activation and quality control of selected LAB strains that has resulted in highly functional and viable LAB culture strains employed for fermentation research. The adaptation and recommendation of a protocol such as this for use in the dairy and food industry&#160;will help to ensure LAB viability and functionality for many applications.

The quality control assessment of lactic acid bacteria (LAB) cultures has been confirmed as an effective way to enhance the viability and functionality of LAB strains for fermentation procedures. To buttress this assertion, we developed a protocol that elucidates how LAB cultures are activated and cultivated for fermentation and bioprocessing procedures.

Lactic acid bacteria (LAB) are essential dairy starter cultures that are significantly employed for the manufacture of fermented dairy products such as ...

A Deferred Growth Inhibition Assay to Evaluate the Effect of Bacteria-Derived Antimicrobial Compounds

Source: Moran, J. C. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/54437">Deferred Growth Inhibition Assay to Quantify the Effect of Bacteria-derived Antimicrobials on Competition.</a> J. Vis. Exp.&#160;(2016)
This video showcases the deferred growth inhibition assay, used to investigate how antimicrobial compounds produced by one type of bacteria affect another competing bacterial strain.

The following method is adapted&#160;to test competitor strains with inhibitor-producing strains.
NOTE: This protocol involves aerosol generation with ...

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

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