This publication was made possible by grant number NC.X-267-5-12-170-1 from the National Institute of Food and Agriculture (NIFA) and in part by NIZO Food Research BV, The Netherlands, Jarrow Formulas, USA, and the Department of Family and Consumer Sciences and the Agriculture Research Station at North Carolina Agriculture and Technical State University (Greensboro, NC, USA 27411). This work was also supported, in part, by 1890 Capacity Building Program grant no. (2020-38821-31113/project accession no. 021765). This work was also partially supported by the Bulgarian Ministry of Education and Science under the National Research Programme &#8216;Healthy Foods for a Strong Bio-Economy and Quality of Life&#8217; approved by DCM # 577 / 17.08.2018.

1. General materials and methods
<ol>
	<li>Source of Lactobacillus delbrueckii subsp. bulgaricus
	<ol>
		<li>Obtain L. bulgaricus strains from reliable sources. 
		NOTE: In this study, a total of five (5) L. bulgaricus strains were used in the quality control study (Table 1). Two strains of freeze-dried L. bulgaricus for the industrial production of fermented milk products were provided by Dr. Albert Krastanov, Department of Biotechnology at the University of Food Technologies, Plovdiv, Bulgaria. Two strains isolated from commercial yogurt products available in the US market were obtained from the -80 °C stock of the Food Microbiology and Biotechnology laboratory at North Carolina A&amp;T State University and one freeze-dried strain was supplied by a vendor C. All LAB strains were kept at -80 °C until further use. Another bacterial strain (Limosilactobacillus reuteri) was obtained from the -80 °C stock of the Food Microbiology and Biotechnology laboratory at North Carolina A&amp;T State University was also evaluated.</li>
	</ol>
	</li>
	<li>Standard L. MRS fermentation broth medium
	<ol>
		<li>Prepare deMan, Rogosa Sharpe (MRS) medium by completely dissolving 55 g of MRS, and 0.5 g of L-Cysteine in 1 L of deionized water (DW).</li>
		<li>Dispense 7 mL and 2 mL of the prepared MRS solution into test tubes, respectively, autoclave at 121 °C for 15 min, and then cool at room temperature (RT).</li>
	</ol>
	</li>
	<li>MRS agar medium
	<ol>
		<li>Prepare MRS agar medium by completely dissolving 55 g of MRS and 0.5 g of L-Cysteine in 1 L of DW. Add agar powder (15 g), sterilize the agar medium at 121 °C for 15 min, and then cool it in a water bath.</li>
		<li>Pour all freshly-prepared media into sterile Petri dishes and store them at 4 °C until further needed.</li>
	</ol>
	</li>
	<li>Modified reinforced clostridial medium-pyruvate (mRCM-PYR)
	<ol>
		<li>Optimize a reinforced clostridial medium according to Oyeniran et al.13 and Nwamaioha and Ibrahim18, for selectivity and accurate enumeration of L. bulgaricus by dissolving 10 g of peptone #3, 10 g of beef extract, 5 g of yeast extract, 5 g of sodium chloride, 3 g of sodium acetate, 2 g of potassium phosphate dibasic, 0.1 g of uracil, 0.25 g of calcium chloride, 5 g of dextrose, 5 g of fructose, 10 g of maltose, 2 g of sodium pyruvate, 0.2% Tween 80, and 0.5 g of L- Cysteine in 1 L of DW.</li>
		<li>Adjust the final pH (6.0 ± 0.2) of the solution using 6 N HCl before the addition of 0.008% aniline blue and 15 g of agar. Autoclave the medium at 121 °C for 15 min, cool in a water bath, and pour into sterile Petri dishes. Store all freshly-prepared media in sterile Petri dishes at 4 °C until needed.</li>
	</ol>
	</li>
	<li>Glycerol stock of LAB cultures
	<ol>
		<li>Prepare glycerol stocks (50% glycerol) by diluting 100% glycerol in the same volume of DW and sterilize at 100 °C for 30 min using a dry sterilization cycle. Cool the glycerol stocks to RT and aseptically store them for further use.</li>
		<li>Use bacterial growth (a single colony of L. bulgaricus obtained using the developed QC protocol) from overnight cultures.</li>
		<li>Pipette 500 µL of the overnight cultures into 50% glycerol in a 2 mL centrifuge tube and gently mix them together. Freeze and store the glycerol stock containing the LAB cultures at an ultra-low temperature of -80 °C until needed. 
		​NOTE: A graphical scheme of the protocol for the quality control and activation of LAB cultures is shown in Figure 1.</li>
	</ol>
	</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>No</td>
			<td>Product Code</td>
			<td>Sample</td>
			<td>Source</td>
			<td>Bacterial Composition as labeled1</td>
		</tr>
		<tr>
			<td>1</td>
			<td>S9</td>
			<td>Pure Industrial Strain</td>
			<td>Bulgaria</td>
			<td>Lb. bulgaricus</td>
		</tr>
		<tr>
			<td>2</td>
			<td>LB6</td>
			<td>Pure Industrial Strain</td>
			<td>Bulgaria</td>
			<td>Lb. bulgaricus,</td>
		</tr>
		<tr>
			<td>3</td>
			<td>ATCC 11842</td>
			<td>Pure Industrial Strain</td>
			<td>ATCC</td>
			<td>Lb. bulgaricus</td>
		</tr>
		<tr>
			<td>4</td>
			<td>DAW</td>
			<td>Yogurt</td>
			<td>USA</td>
			<td>Lb. bulgaricus, other live culture</td>
		</tr>
		<tr>
			<td>5</td>
			<td>E22</td>
			<td>Yogurt</td>
			<td>USA</td>
			<td>Lb. bulgaricus, other live culture</td>
		</tr>
		<tr>
			<td>6</td>
			<td>Reuteri</td>
			<td>Yogurt</td>
			<td>USA</td>
			<td>Limosilactobacillus reuteri</td>
		</tr>
		<tr>
			<td colspan="5">1Lb. =Lactobacillus</td>
		</tr>
	</tbody>
</table>
Table 1: Probiotic strains. The table lists the probiotic strains used in this study.
2. Protocol for the activation and quality control of LAB cultures
<ol>
	<li>Take the prepared glycerol stock of LAB strains (in 2 mL centrifuge tubes) from the -80 °C ultra-low freezer, and do not allow them to thaw before use.</li>
	<li>Clean and disinfect the opening of the centrifuge tubes with 70% alcohol, and gently vortex before use.</li>
	<li>Pipette about 250 µL (0.25 mL) of the stock LAB culture from the centrifuge tubes into fresh 2 mL MRS test tubes.</li>
	<li>Gently vortex, parafilm the test tubes, and anaerobically incubate them overnight at 42 °C for 12-16 h.</li>
	<li>Take about 500 µL (0.5 mL) from the overnight grown cultures from the 2 mL MRS test tubes into fresh 7 mL MRS test tubes, vortex, and anaerobically incubate them overnight at 42 °C for 12-16 h.</li>
	<li>Assess the microbial growth by measuring the optical density (OD) or growth of the cultures at 610 nm with a UV- visible spectrophotometer and record acceptable results between 0.7 and 0.9.</li>
	<li>Streak the overnight cultures from the 7 mL MRS tubes onto MRS and MRCM-PYR agar plates and incubate them anaerobically for 72 h at 42 °C.</li>
	<li>Pick isolated colonies from the agar plates, transfer them into fresh 7 mL MRS test tubes, gently vortex, and anaerobically incubate them overnight at 42 °C for 12-16 h.</li>
	<li>Store the agar plates containing the isolated strains at 4 °C in the refrigerator for a week.</li>
	<li>Measure and confirm the OD (between 0.7 and 0.9) from the 7 mL MRS test tubes of the LAB cultures isolated from the streaked plates at 610 nm and use them as working cultures for all related experiments.</li>
	<li>Perform ten-fold dilutions (serially dilute) of the grown LAB cultures from the final 7 mL MRS test tubes using 9 mL of peptone water (a physiological buffer) to obtain a 1:10 ratio.</li>
	<li>Finally, take about 250 µL (0.25 mL) from appropriate serial dilutions for all fermentation experiments.</li>
	<li>Activate the broth containing strains (250 µL) from step 2.12 by transferring them into fresh 7 mL MRS broth and incubating them anaerobically at 42 °C for 16 h.</li>
	<li>Continue by repeating steps 2.6-2.12 to ensure viable and superior cell growth from LAB cultures. 
	NOTE: All L. bulgaricus strains were activated in the freshly prepared MRS broth and were then incubated anaerobically at 42 °C for 16 h in order to reach an optical density (OD610 nm) of bacterial growth between 0.7 and 0.9. Bacterial growth was measured with a UV-visible spectrophotometer at 610 nm. The pH values of the overnight cultures were in the range of 3.5 to 5.3 as a result of the production of lactic acid, an organic end product of LAB fermentation. Safety procedures such as proper airflow circulation in the biosafety hood and avoiding burns during the use of the bunsen burner were adhered to and observed.</li>
</ol>
<img alt="LAB Growth: Fermentation diagram showing MRS broth inoculation, incubation, optical density measurement." class="xfigimg" src="/files/ftp_upload/63314/63314fig01.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1: A graphical scheme of the protocol for the activation of lactic acid bacteria (LAB) cultures. The scheme provides details and the basic instruments required for the handling and activation of LAB culture strains. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63314/63314fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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</ol>

The authors have nothing to disclose.

The results of all strains evaluated with the quality control protocol and without the use of the protocol were the same, and as such, results linked to only strains (S9, and LB6) were presented. The activated LAB strains had superior cell growth that was characterized by a high intensity of cell biomass, therefore, causing a turbid appearance of the MRS fermentative broth in the test tube11. The observed cell growth after culture activation was evident between 12 h and 16 h at an anaerobic fermentative temperature of 42 &#176;C. It was also confirmed that the LAB strains activated based on this protocol continuously had their colony morphologies transformed as a result of the multi sub-culturing procedure12 with discrete colonies visible along the lines of streaking. The control experiment did not employ this standard protocol and was only subcultured once; it, however, did not also show intense turbidity in the fermentative broth, although growth was still within the accepted range of 0.7-0.9 at OD610 nm. It was also noteworthy that streaking the control cultures on the agar plates and anaerobically incubating them under the same conditions as the standard procedure only depicted smaller and fewer colonies as compared to those observed in the standard protocol13.
This could be attributed to the weak and non-viable characteristics of the LAB cells. Consequently, the concept of sub-culturing LAB strains multiple times and streaking on agar plates, thus, results in the generation of new cells through cell division and exchange of genetic matter in the bacteria. This enhances cellular and metabolic functionality and promotes the growth of cells during fermentation operations14. In addition, stressed and injured LAB cells are also recovered based on the steady-state of growth conditions (nutritional requirements, pH, and temperature) that enable them to easily adapt and regain full cell and metabolic functionality6. Moreover, strict adherence to this protocol limits contamination of cell cultures to a greater extent, thereby ensuring only pure and superior cell colonies are isolated for fermentation purposes. Thus, repeated streaking and recovery of LAB cell cultures from agar plates provided an enhanced cell wall that was well suited for metabolic activities such as for LAB fermentation operations15. Furthermore, the study on the growth of L. reuteri based on the developed quality control protocol (Figure 3) confirms how subculturing LAB via colony to culture method yields a stable steady growth at the optimum growth range (0.9-1.0 OD610 nm) over a period as compared to subculturing directly from culture to culture (conventional method), which resulted in a sharp growth from 0.75 to 0.9 at OD610 nm. It was also evident that the QC protocol, over time, consistently yielded a stable bacteria growth pattern, which had a standard deviation (SD) of 0.1. The uneven growth of L. reuteri, however, had its SD greater than 0.1 due to the uneven growth of the bacterial cells. This uneven growth pattern, as already stated, was attributed to weak and injured cells; thus metabolic functionality of the cells decreased during the fermentation period14. The current study was also confirmed by a previous one by Ahmed et al.16 whereby, in their study, L. reuteri was propagated in trypticase peptone yeast extract (TPY) agar medium for 8 weeks at 37 &#176;C. A contributory factor for the steady growth of L. reuteri was attributed to the method of cell cultivation and quality control technique that generated new and superiorly active cultures even after several subculturing activities during the study period.
The critical steps in this protocol include always employing new and less than a day old fermentative cultures and always having the fermentative broth or growth medium newly prepared before commencement of fermentation, and not using stored growth medium as the strength and potency of the medium decreases over an extended period of storage. In the case of using stock cultures from either a -80 &#176;C&#160;or -20 &#176;C, the cultures should be aseptically handled. Furthermore, if stock cultures are observed to be weak due to cold shock from the freezer, resuscitation is required as follows. Only low concentrations (about 100 &#181;L) of the stock cultures should be inoculated in the supplemented fermentative broth (MRS) of about 2 mL for pre-anaerobic incubation for about 6 h before additional supplemented MRS of about 5-6 mL is added for final anaerobic incubation for about 16 h17. In the event of using lyophilized or freeze-dried cultures, it is highly recommended to recover the freeze-dried cultures in 3 mL of skimmed fluid milk and anaerobically incubate them for about 4-5 h before inoculating them in the supplemented fermentative broth such as MRS. These processes are key in ensuring LAB cell viability at all times13.
Another critical step is to ensure cell growth of fermentative cultures is always within the growth range of 0.7-0.9, which is usually at OD610nm. This is important as that growth range is recommended as the appropriate log or exponential phase of the bacterial growth curve17. Any optical density (OD) value above 1, is inaccurate as this confirms the fermentative cultures have reached their death phase with most dead cells and accumulation of toxic cellular waste predominantly present in the fermentative medium. In the event of reaching an OD value above 1 (OD610nm &#62; 1)., it is recommended to dilute the cultures 2 or 3-fold or more until it reaches an OD value below 1 (OD610nm &#60; 1)18. Another point of concern is to appropriately anaerobically incubate the streaked plates with the fermentative cultures. Although most LAB are facultative anaerobes, enhanced growth is limited in the presence of oxygen; hence the preference for anaerobic conditions is required to promote efficient growth. It is recommended to use a CO2 chamber or an improvised vessel that could generate CO2 during the incubation period at 42 &#176;C. For best cell growth results on incubated plates, 72 h is recommended for optimum results18.
There are, however, no limitations of this standard procedure, as strict adherence to the steps will ensure viable and pure LAB cells for fermentative procedures. The significance of this standard protocol is based on its proactive approach to ensuring probiotic cell viability, especially for fermentative cultures. It serves as a quality control mechanism that seeks to eliminate contamination and ultimately promote strong and highly viable cultures that could produce desirable yields of fermentation under standard conditions. In reference to existing or conventional methods, this standard protocol eliminates the chance of LAB cell viability challenges (due to cold shock, and poor growth medium) it also seeks to ensure the first-time-right concept of having pure and viable probiotic cultures, saves time, and promotes productivity when working with LAB cultures.
The future applications of this technique are enormous, as this protocol could be used in both research laboratories, dairy and food industries, bioprocessing, fermentation, and biotechnology institutes. The added advantage of this protocol is based on its simplistic nature and thus does not require sophisticated use of laboratory equipment. As such, this protocol could be used for demonstration or pedagogical learning in high schools that have a basic laboratory environment to encourage and build the interest of students in Science, Technology, Engineering, and Mathematics (STEM) related fields.

Lactic acid bacteria (LAB) are a group of uniquely diverse bacteria that have industrial potential. Strains belonging to Lactobacillus delbreuckii subsp. bulgaricus and Streptococcus thermophilus are mostly used as dairy starter cultures for fermented dairy food products such as yogurt1. Selected LAB strains are also classified as probiotics as they confer health benefits to humans when dosages are adequately administered2. Lactic acid bacteria are also gram-positive, non-spore-forming, non-respiring but aerotolerant microorganisms that are generally characterized by the production of lactic acid as a key fermentation product. LAB also synthesizes essential metabolites, for example, organic acids, bacteriocins, and other antimicrobial compounds3 that can inhibit a broad spectrum of foodborne pathogens4. Lactic acid, a major end product of carbohydrate catabolism and a by-product of LAB fermentation, is an organic metabolite that possesses antimicrobial properties and is potentially useful for food biopreservation applications3,5,6. Furthermore, the organic acids produced by LAB impart the flavor, texture, and aroma of foods, thus consequently enhancing their overall organoleptic properties5,6. The distinct nutritional requirements of LAB coupled with their ubiquitous nature, ultimately enable the bacteria to easily thrive in different environments such as dairy-based foods, fermented foods, vegetables as well as in the human gut7.
There is a growing demand for starter cultures from LAB for yogurt production and many diverse dairy applications8,9, hence critical attention and established scientific techniques should be adhered to, in LAB strains cultivation, as well as in the activation of both lyophilized and isolated strains as this activity is vital for enhanced fermentation performance. The Food Microbiology and Biotechnology laboratory, therefore, actively engages in suitable technology development geared toward the activation, superior growth, and fermentation characteristic of LAB strains isolated from fermented dairy products as well as from industrial starter cultures employed for yogurt production. Furthermore, it is noteworthy that LAB culture strains industrially produced undergo preservative activities such as freeze-drying and frozen storage, causing cell stress and injury, as a result of the cold shock process they are subjected to10. In limiting, the viability challenges and improving the functionality of LAB strains obtained from either isolated food products or freeze-dried products, it is important to properly activate these cultures as a form of quality control to enhance their fermentative characteristic8. In this study, the objective was to develop an in-house quality control protocol for the activation and growth of L. delbrueckii subsp. bulgaricus culture strains that ultimately promoted viable LAB growth, as well as enhanced the fermentation performance and the metabolic functionality of LAB strains. This protocol could ultimately be adapted (using optimal growth media and appropriate culturing conditions) for the cultivation of other LAB strains for fermentation research, as well as for industrial purposes or bioprocessing operations. This LAB activation and quality control protocol will therefore ensure superior viable dairy starter cultures are obtained and potentially functional for diverse applications in the global dairy and food industry.

<table><tbody><tr><td>Aniline Blue</td><td>Thermo Scientific</td><td>R21526</td><td>25 g</td></tr><tr><td>Beef extract</td><td>Research Products International</td><td>50-197-7509</td><td>500 g</td></tr><tr><td>Yeast extract</td><td>Fisher Scientific</td><td>BP1422-500</td><td>500 g</td></tr><tr><td>Calcium Chloride dihydrate</td><td>Fisher Scientific</td><td>C79-500</td><td>500 g</td></tr><tr><td>Dextrose Anhydrous</td><td>Fisher Scientific</td><td>BP350500</td><td>500 g</td></tr><tr><td>D-Fructose</td><td>ACROS Organics</td><td>AC161355000</td><td>500 g</td></tr><tr><td>Difco agar powder</td><td>Difco</td><td>DF0812-07-1</td><td>2 kg</td></tr><tr><td>TPY agar</td><td>Difco</td><td>211921</td><td>500 g</td></tr><tr><td>Eppendorf microcentrifuge tube (Snap-Cap Microcentrifuge Safe-Lock)</td><td>Fisher Scientific</td><td>05-402-12</td><td>2 mL</td></tr><tr><td>Glycerol</td><td>Thermo Scientific</td><td>PI17904</td><td>500 mL</td></tr><tr><td>Infrared CO2 Incubator</td><td>Forma Scientific</td><td></td><td></td></tr><tr><td>Lactobacillus delbrueckii subsp. bulgaricus</td><td>American Type Culture Collection (ATCC)</td><td>ATCC 11842</td><td></td></tr><tr><td>Lactobacillus delbrueckii subsp. bulgaricus</td><td>Bulgaria</td><td>S9</td><td></td></tr><tr><td>Lactobacillus delbrueckii subsp. bulgaricus</td><td>Bulgaria</td><td>LB6</td><td></td></tr><tr><td>Lactobacillus delbrueckii subsp. bulgaricus</td><td>Food Microbiology and Biotechnology Laboratory (NCATSU)</td><td>DAW</td><td></td></tr><tr><td>Lactobacillus delbrueckii subsp. bulgaricus</td><td>Food Microbiology and Biotechnology Laboratory (NCATSU)</td><td>E22</td><td></td></tr><tr><td>Limosilactobacillus reuteri</td><td>Biogai, Raleigh / Food Microbiology and Biotechnology Laboratory (NCATSU)</td><td>RD2</td><td></td></tr><tr><td>L-Cysteine hydrochloride monohydrate</td><td>Sigma-Aldrich</td><td>C6852-25G</td><td>25 g</td></tr><tr><td>Maltose monohydrate</td><td>Fisher Scientific</td><td>M75-100</td><td>100 g</td></tr><tr><td>MRS broth</td><td>Neogen</td><td>50-201-5691</td><td>5 kg</td></tr><tr><td>Peptone No. 3</td><td>Hach</td><td>50-199-6719</td><td>500 g</td></tr><tr><td>Potassium phosphate dibasic (K2HPO4)</td><td>Research Products International</td><td>50-712-761</td><td>500 g</td></tr><tr><td>Sodium acetate trihydrate</td><td>Fisher Scientific</td><td>S220-1</td><td>1 kg</td></tr><tr><td>Sodium chloride</td><td>Fisher Scientific</td><td>BP358-1</td><td>1 kg</td></tr><tr><td>Sodium pyruvate</td><td>Fisher Scientific</td><td>BP356-100</td><td>100 g</td></tr><tr><td>Test Tubes with Rubber-Lined Screw Caps</td><td>Fisher Scientific</td><td>FB70125150</td><td>25 x 150 mm</td></tr><tr><td>Tween 80</td><td>Fisher Scientific</td><td>T164-500</td><td>500 mL</td></tr><tr><td>Ultra low freezer</td><td>So-Low</td><td></td><td></td></tr><tr><td>Uracil</td><td>ACROS Organics</td><td>AC157301000</td><td>100 g</td></tr><tr><td>UV- visible spectrophotometer</td><td>Thermo Fisher Scientific</td><td>Evolution 201</td><td></td></tr><tr><td>Vortex Genie 2</td><td>Fisher Scientific</td><td></td><td></td></tr><tr><td>Yeast extract</td><td>Fisher Scientific</td><td>BP1422-500</td><td>500 g</td></tr><tr><td>Ethanol</td><td>Fisher Scientific</td><td>T08204K7</td><td>4 L</td></tr><tr><td>Hydrochloric Acid (6N (Certified), Fisher Chemical)</td><td>Fisher Scientific</td><td>&amp;nbsp;SA56-500</td><td>500 mL</td></tr></tbody></table>

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.

Cell growth of the evaluated LAB strains cultivated with the quality control protocol was significantly different (P &lt; 0.05) than the strains cultivated without this standard protocol. The QC protocol for both L. bulgaricus and L. reuteri employed a multi-subculturing approach (subculturing three times before streaking on agar plates), whereas the control procedure had subculturing done only once with all other conditions kept constant. The colony growth was also higher and well defined on the growth media plates that were cultivated using this standard QC protocol. This confirmed the effectiveness of the protocol in ensuring enhanced LAB growth through cell division in the fermentative broth, therefore, aiding enhanced cell metabolism in the LAB strains. The growth of two strains cultivated with and without the developed quality control protocol is shown in Figure 2. The strains were streaked onto agar plates after intense growth (0.7-0.9 at OD610 nm) was observed and measured. The plates were anaerobically incubated at 42 °C for 72 h after which discrete colonies were observed along the lines of streaking. Another study (Figure 3) confirmed the efficiency of the quality control protocol on the growth of Lactobacillus reuteri cultured in TPY medium and, incubated at 37 °C, whereby colonies generated by the QC protocol technique confirmed a stable steady-state growth (averagely 0.9 at OD610 nm) based on historical data. A control method of LAB activation without multiple sub-culturing that did not employ the developed QC technique resulted in an uneven growth pattern (0.9-0.75 at OD610 nm) of L. reuteri.
<img alt="Lb. bulgaricus strains growth comparison; petri dish results; quality control; bacterial culture." class="xfigimg" src="/files/ftp_upload/63314/63314fig02.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2: Colonial morphology of growth of L. bulgaricus strains cultivated with and without the developed quality control protocol. The strains were streaked in triplicates and were anaerobically incubated at 42 °C for 72 h. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63314/63314fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Bacterial growth over years line chart; QC vs. conventional protocols; OD measurements; error bars." class="xfigimg" src="/files/ftp_upload/63314/63314fig03.jpg" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
Figure 3: Behavior of L. reuteri over a period after growth in TPY medium at 37 °C using the standardized QC protocol and the conventional method of bacterial culturing (P &lt; 0.05).  The growth of L. reuteri was monitored periodically for a month and was aligned with historical data from previous years based on culture activation with the standard QC protocol. The error bars indicate the standard deviation of the OD values. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63314/63314fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about The Cultivation, Growth, and Viability of Lactic Acid Bacteria: A Quality Control Perspective at JoVE.com

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

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 ...

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Nanyang Technological University

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Biology

7.9K Views.  North Carolina A&T State University, Greensboro, NC, USA. Ons protocol is belangrijk omdat het zorgt voor zuiverheid als een kwaliteitscontrole. Het bevordert ook de prestaties van ernstige fermentatie en de consistentie van de opbrengst bij de fermentatie van melkzuurbacteriën. Het belangrijkste voordeel van deze techniek is dat het de fermentatieprestaties van melkzuurbacteriën kan verbeteren via celzuiverheid, levensvatbaarheid en functionaliteit.Deze techniek is gebruiksvriendelijk en kan gemakkelijk door iedereen worden uitgevoerd vanw...

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

Methods for Facilitating Microbial Growth on Pulp Mill Waste Streams and Characterization of the Biodegradation Potential of Cultured Microbes

The kraft process is applied to wood chips for separation of lignin from the polysaccharides within lignocellulose for pulp that will produce a high quality paper. Black liquor is a pulping waste generated by the kraft process that has potential for downstream bioconversion. However, the recalcitrant nature of the lignocellulose resources, its chemical derivatives that constitute the majority of available organic carbon within black liquor, and its basic pH present challenges to microbial biodegradation of this waste material. Methods for the collection and modification of black liquor for microbial growth are aimed at utilization of this pulp waste to convert the lignin, organic acids, and polysaccharide degradation byproducts into valuable chemicals. The lignocellulose extraction techniques presented provide a reproducible method for preparation of lignocellulose growth substrates for understanding metabolic capacities of cultured microorganisms. Use of gas chromatography-mass spectrometry enables the identification and quantification of the fermentation products resulting from the growth of microorganisms on pulping waste. These methods when used together can facilitate the determination of the metabolic activity of microorganisms with potential to produce fermentation products that would provide greater value to the pulping system and reduce effluent waste, thereby increasing potential paper milling profits and offering additional uses for black liquor.

Industrial wastes can be collected and modified to analyze microbial growth. Lignocellulose extraction techniques provide components to analyze specific biodegradation ability. Gas chromatography-mass spectrometry identifies fermentation products of microorganisms grown on pulping waste. These methods determine the metabolic capacity of microorganisms to degrade pulping waste.

The kraft process is applied to wood chips for separation of lignin from the polysaccharides within lignocellulose for pulp that will produce a high ...

Environment

Au-Interaction of Slp1 Polymers and Monolayer from Lysinibacillus sphaericus JG-B53 - QCM-D, ICP-MS and AFM as Tools for Biomolecule-metal Studies

In this publication the gold sorption behavior of surface layer (S-layer) proteins (Slp1) of Lysinibacillus sphaericus JG-B53 is described. These biomolecules arrange in paracrystalline two-dimensional arrays on surfaces, bind metals, and are thus interesting for several biotechnical applications, such as biosorptive materials for the removal or recovery of different elements from the environment and industrial processes. The deposition of Au(0) nanoparticles on S-layers, either by S-layer directed synthesis 1 or adsorption of nanoparticles, opens new possibilities for diverse sensory applications. Although numerous studies have described the biosorptive properties of S-layers 2-5, a deeper understanding of protein-protein and protein-metal interaction still remains challenging. In the following study, inductively coupled mass spectrometry (ICP-MS) was used for the detection of metal sorption by suspended S-layers. This was correlated to measurements of quartz crystal microbalance with dissipation monitoring (QCM-D), which allows the online detection of proteinaceous monolayer formation and metal deposition, and thus, a more detailed understanding on metal binding.
The ICP-MS results indicated that the binding of Au(III) to the suspended S-layer polymers is pH dependent. The maximum binding of Au(III) was obtained at pH 4.0. The QCM-D investigations enabled the detection of Au(III) sorption as well as the deposition of Au(0)-NPs in real-time during the in situ experiments. Further, this method allowed studying the influence of metal binding on the protein lattice stability of Slp1. Structural properties and protein layer stability could be visualized directly after QCM-D experiment using atomic force microscopy (AFM). In conclusion, the combination of these different methods provides a deeper understanding of metal binding by bacterial S-layer proteins in suspension or as monolayers on either bacterial cells or recrystallized surfaces.

Au-Interaction of Slp1 Polymers and Monolayer from Lysinibacillus sphaericus JG-B53 - QCM-D, ICP-MS and AFM as Tools for Biomolecule-metal Studies

To obtain basic information on the sorption and recycling of gold from aqueous systems the interaction of Au(III) and Au(0) nanoparticles on S-layer proteins were investigated. The sorption of protein polymers was investigated by ICP-MS and that of proteinaceous monolayers by QCM-D. Subsequent AFM enables the imaging of the nanostructures.

In this publication the gold sorption behavior of surface layer (S-layer) proteins (Slp1) of Lysinibacillus sphaericus JG-B53 is described. These ...

Chemistry

A Gnotobiotic System for Studying Microbiome Assembly in the Phyllosphere and in Vegetable Fermentation

The phyllosphere, the above ground portion of the plant that can be colonized by microbes, is a useful model system to identify processes of microbial community assembly. This protocol outlines a system for studying microbial community dynamics in the phyllosphere of Napa cabbage plants. It describes how to grow germ-free plants in test tubes with a calcined clay and nutrient broth substrate. Inoculation of germ-free plants with specific microbial cultures provides opportunities to measure microbial growth and community dynamics in the phyllosphere. Through the use of sterile vegetable extract produced from cabbages shifts in microbial communities that occur during fermentation can also be assessed. This system is relatively simple and inexpensive to set up in the lab and can be used to address key ecological questions in microbial community assembly. It also provides opportunities to understand how phyllosphere community composition can impact the microbial diversity and quality of vegetable fermentations. This approach for developing gnotobiotic cabbage phyllosphere communities could be applied to other wild and agricultural plant species.

A method of growing germ-free Napa cabbages has been developed which enables researchers to evaluate how single microbial species or multispecies microbial communities interact on cabbage leaf surfaces. A sterile vegetable extract is also presented which can be used to measure shifts in community composition during vegetable fermentation.

The phyllosphere, the above ground portion of the plant that can be colonized by microbes, is a useful model system to identify processes of microbial ...

Medium Preparation for the Cultivation of Microorganisms under Strictly Anaerobic/Anoxic Conditions

In contrast to aerobic organisms, strictly anaerobic microorganisms require the absence of oxygen and usually a low redox potential to initiate growth. As oxygen is ubiquitous in air, retaining O2-free conditions during all steps of cultivation is challenging but a prerequisite for anaerobic culturing. The protocol presented here demonstrates the successful cultivation of an anaerobic mixed culture derived from a biogas plant using a simple and inexpensive method. A precise description of the entire anoxic culturing process is given including media preparation, filling of cultivation flasks, supplementation with redox indicator and reducing agents to provide low redox potentials as well as exchanging the headspace to keep media free from oxygen. Furthermore, a detailed overview of aseptically inoculating gas tight serum flasks (by using sterile syringes and needles) and suitable incubation conditions is provided. The present protocol further deals with gas and liquid sampling for subsequent analyses regarding gas composition and volatile fatty acid concentrations using gas chromatography (GC) and high performance liquid chromatography (HPLC), respectively, and the calculation of biogas and methane yield considering the ideal gas law.

As obligate anaerobic organisms are unable to grow upon oxygen exposure, the use of anaerobic culturing techniques is indispensable. Here, we demonstrate a simple and effective method to cultivate a mixed culture derived from a biogas plant from media preparation to gas and volatile fatty acid quantification.

In contrast to aerobic organisms, strictly anaerobic microorganisms require the absence of oxygen and usually a low redox potential to initiate growth. As ...

Biochemistry

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. ...

Bioengineering

Microbial Control and Monitoring Strategies for Cleanroom Environments

Microbial Control and Monitoring Strategies for Cleanroom Environments and Cellular Therapies

A well-validated and holistic program that incorporates robust gowning, cleaning, environmental monitoring, and personnel monitoring measures is critical for minimizing the microbial bioburden in cellular therapy manufacturing suites and the corresponding testing laboratories to ensure that the facilities are operating in a state of control. Ensuring product safety via quality control measures, such as sterility testing, is a regulatory requirement for both minimally manipulated (section 361) and more than minimally manipulated (section 351) human cells, tissues, and cellular and tissue-based products (HCT/Ps). In this video, we provide a stepwise guide for how to develop and incorporate the best aseptic practices for operating in a cleanroom environment, including gowning, cleaning, staging of materials, environmental monitoring, process monitoring, and product sterility testing using direct inoculation, provided by the United States Pharmacopeia (USP&lt;71&gt;) and the National Institutes of Health (NIH) Alternative Sterility Testing Method. This protocol is intended as a reference guide for establishments expected to meet current good tissue practices (cGTP) and current good manufacturing practices (cGMP).

Author Spotlight: Microbial Control and Monitoring Strategies for Cleanroom Environments and Cellular Therapies  

The protocol summarizes the best practices to minimize microbial bioburden in a cleanroom environment and includes strategies such as environmental monitoring, process monitoring, and product sterility testing. It is relevant for manufacturing and testing facilities that are required to meet current good tissue practice standards and current good manufacturing practice standards.

A well-validated and holistic program that incorporates robust gowning, cleaning, environmental monitoring, and personnel monitoring measures is critical ...

Isolation, Purification, and Identification of Bacitracin-producing Bacillus licheniformis from Fresh Feces of Healthy Pigs

Bacillus licheniformis and bacitracin have a huge application market and value in the fields of medicine, chemistry, aquaculture, agricultural, and sideline products. Therefore, the selection of B. licheniformis with high production of bacitracin is of great importance. In this experimental protocol, Bacillus with a high yield of bacitracin was isolated, purified, and identified from the fresh feces of healthy pigs. The inhibitory effect of secondary metabolite bacitracin on Micrococcus luteus was also tested. Thin-layer chromatography and high-performance liquid chromatography were used for the qualitative and quantitative detection of bacitracin. The physiological and biochemical characteristics of B. licheniformis were determined by relevant kits. The phylogenetic relationships of B. licheniformis were determined and constructed using gene sequence detection. This protocol describes and introduces the standard isolation, purification, and identification process of B. licheniformis from animal fresh feces from multiple perspectives, providing a method for the large-scale utilization of B. licheniformis and bacitracin in factories.

Isolation, Purification, and Identification of Bacitracin-producing Bacillus licheniformis from Fresh Feces of Healthy Pigs

This protocol provides a complete and comprehensive description of the detailed process for the isolation, purification, and identification of bacitracin-producing Bacillus licheniformis from healthy pig feces.

Bacillus licheniformis and bacitracin have a huge application market and value in the fields of medicine, chemistry, aquaculture, agricultural, and ...

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 ...

A Method to Assess Bacteriocin Effects on the Gut Microbiota of Mice

Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the factors that contribute to maintaining the population balance. However, there are limited available methodologies to evaluate these factors. Bacteriocins are antimicrobial peptides produced by many bacteria that may confer a competitive advantage for food acquisition and/or niche establishment. Many probiotic lactic acid bacteria (LAB) strains have great potential to promote human and animal health by preventing the growth of pathogens. They can also be used for immuno-modulation, as they produce bacteriocins. However, the antagonistic activity of bacteriocins is normally determined by laboratory bioassays under well-defined but over-simplified conditions compared to the complex gut environment in humans and animals, where bacteria face multifactorial influences from the host and hundreds of microbial species sharing the same niche. This work describes a complete and efficient procedure to assess the effect of a variety of bacteriocins with different target specificities in a murine system. Changes in the microbiota composition during the bacteriocin treatment are monitored using compositional 16S rDNA sequencing. Our approach uses both the bacteriocin producers and their isogenic non-bacteriocin-producing mutants, the latter giving the ability to distinguish bacteriocin-related from non-bacteriocin-related modifications of the microbiota. The fecal DNA extraction and 16S rDNA sequencing methods are consistent and, together with the bioinformatics, constitute a powerful procedure to find faint changes in the bacterial profiles and to establish correlations, in terms of cholesterol and triglyceride concentration, between bacterial populations and health markers. Our protocol is generic and can thus be used to study other compounds or nutrients with the potential to alter the host microbiota composition, either when studying toxicity or beneficial effects.

Bacteriocins are believed to play a key role in defining microbial diversity in different ecological niches. Here, we describe an efficient procedure to assess how bacteriocins affect gut microbiota composition in an animal model.

Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the ...

Quantitative Analysis of Microbial Autoaggregation Using Imaging Flow Cytometry

Imaging Flow Cytometry to Study Microbial Autoaggregation

Beneficial and probiotic bacteria play essential roles in their hosts, providing various health benefits, including immunity to infectious diseases. The Lactobacillaceae family consists of Gram-positive bacteria with confirmed probiotic properties. This study utilizes Lactobacillaceae species as a model to demonstrate the effectiveness of single-cell high throughput analysis in studying cellular aggregation. The focus is on analyzing the response of these beneficial species to simple carbohydrates from the diet.
The study showcases how Imaging Flow Cytometry (IFC) can overcome the fundamental differences in the assembly of probiotic bacteria in the presence and absence of carbohydrates. IFC combines the power and speed of conventional flow cytometry with the spatial resolution of microscopy, enabling high-rate complex morphometric measurements in a phenotypically defined manner across a library of beneficial bacterial strains and conditions. This protocol provides insights into the autoaggregation of Lactobacillaceae species and sheds light on their response to dietary carbohydrates, contributing to understanding the mechanisms behind the beneficial effects of these probiotic bacteria.

Author Spotlight: Advancing Research in Microbial Autoaggregation Using Imaging Flow Cytometry

This protocol describes a quantitative approach to measure microbial autoaggregation using imaging flow cytometry.

Beneficial and probiotic bacteria play essential roles in their hosts, providing various health benefits, including immunity to infectious diseases. The ...

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

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