This research was financially supported by the earmarked fund for Jiangsu Natural Science Fund (BK20170185), Project from Jiangsu Fisheries Administrator (Y2017-30), National Natural Science Foundation of China (NFSC31801575), China Agricultures Research System (CARS-45-26), national first-class discipline program of Food Science and Technology (JUFSTR20180201), and Yi Tong-Jiangsu Postdoctoral Program.

1. Sample preparation
<ol>
	<li>Thaw frozen sturgeon meat under flowing water below 20 &#176;C. Then, clean, skin off and cut the fish into cubes (2 cm &#215; 2 cm &#215; 1.5 cm). 
	NOTE: Raw material could be fresh or frozen sturgeon meat.</li>
	<li>Mix fish cubes with 6-12% (w/v) salt solution at a rate of 1:1 (fish cube mass to solution volume), at 10 &#176;C for 1-3 h.</li>
	<li>Put the marinated fish cubes onto a stainless-steel gridding plate and dry fish cubes at 40-60 &#176;C for about 6-10 h by hot air, or dry the fish cubes in vacuum, until the water content decreases to 50-60%.</li>
	<li>Mix dried fish cubes with the flavor solution inoculating 0.4-1.6% (w/w, starter culture mass to fish cube mass) S. cerevisiae at a rate of 1:1 (fish cube mass to solution volume). Seal the mixture in a container and ferment at 25-35 &#176;C for 6-10 h. 
	NOTE: The fermentation must be performed under anaerobic environment.
	<ol>
		<li>Prepare the flavor solution to include 25% spices liquid, 40% yellow wine, 7% white wine, 25% cane sugar, 2% monosodium glutamate, and 1% vinegar in mass fraction.</li>
		<li>Prepare the spice liquid is as follows. Mix spices including 1.2 units of scallions, 1 unit of ginger, 0.6 units of star anise, 0.6 units of fennel, 0.3 units of green tea and 0.3 units of pepper with 40 units of water and boil for 0.5 h. Collect the filtrate. Mix the residue with another 40 units of water and boil for 0.5 h again. Combine filtrates of the two boils and make up to 100 units using boiled water. 
		NOTE: It is a must to pause the protocol here until the spice liquid has cooled to the room temperature before the addition of starter culture. This pause could avoid inactivation of yeast by heat.</li>
	</ol>
	</li>
	<li>Seal fermented fish cubes in vacuum package with marinating and fermenting solutions at a rate of 11-13:1 (fish cube mass to solution volume). The vacuum strength, sealing time and cooling time are 0.085 MPa, 3.6 s and 5.5 s, respectively. 
	NOTE: The vacuum package material is polyethylene terephthalate/cast polypropylene (PET/CPP). The package weight is 50-80 g.</li>
	<li>Sterilize packed fish cubes before storage and sale. The sterilization F-value should be more than 4.5 min. The sterilization temperature and time are 115-121 &#176;C and 10-20 min, respectively. Maintain pressure constant at 0.12 MPa for cooling after sterilization. 
	NOTE: When sterilization F-value is 4.5 min and sterilization temperature is set as 121 &#176;C, the sterilization time is 11.4 min in the equipment conditions of this article. Revise the sterilization time when the type of equipment used is different.</li>
</ol>
2. Estimation of shelf life of fermented sturgeon meat product
NOTE: The estimation of shelf life of fermented sturgeon meat product uses accelerated shelf life testing (ASLT) method with Arrhenius model according to the method of Wahyuni et al. with some modifications18.
<ol>
	<li>Store products at 20 &#176;C, 30 &#176;C, 40 &#176;C.</li>
	<li>Predict the shelf life by the Arrhenius model using rancidity rates. Measure rancidity rates by testing acid values (AV) under different temperatures.</li>
	<li>Measure AV on the 0th, 14th, 28th, 42nd, 56th, 70th days.</li>
</ol>
3. Chemical analysis
<ol>
	<li>Determination of salt content 
	NOTE: We measure salt content according to the method proposed by Zeng et al.19.
	<ol>
		<li>Accurately weigh 1 g of the sample and add 10 mL of 0.1 M AgNO3 + 10 mL of HNO3.</li>
		<li>Heat the mixture gently on an induction-cooker for 10 min.</li>
		<li>Cool the mixture with running water, and add distilled water (50 mL) and ferric alum indicator (5 mL).</li>
		<li>Titrate the mixture with standard 0.1 M KSCN until the solution turns permanent brownish-red. 
		NOTE: Calculate and express the results as % NaCl.</li>
	</ol>
	</li>
	<li>Determination of moisture content
	<ol>
		<li>Determine moisture content according to the method of Zeng et al.19.</li>
	</ol>
	</li>
	<li>Determination of pH value 
	NOTE: We measure pH value according to the method of Zeng et al.6.
	<ol>
		<li>Homogenize 10 g of the sample with 90 mL of deionized water.</li>
		<li>Measure the pH value with a digital pH meter.</li>
	</ol>
	</li>
</ol>
4. Headspace solid-phase microextraction followed by gas chromatography-mass spectrometry (SPME-GC/MS) analysis
NOTE: We measure flavor according to the method of Gao et al. with some modification17.
<ol>
	<li>Extraction of volatile flavors
	<ol>
		<li>Weigh 2 g of the sample accurately, and then put it into the sample vial.</li>
		<li>Add 2.5 mL of saturated sodium chloride solution and 0.5 M 2,4,6-trimethylpyridine into the sample vial with a rotor, and then close the vial cap.</li>
		<li>Put the sample vial on the magnetic stirrer and stir for 10 min to mix the sample homogenously.</li>
		<li>Insert the fiber into the headspace of the sample vial.</li>
		<li>Adsorb at 60 &#176;C for 30 min, and then desorb at 250 &#176;C for 3 min in the injection port.</li>
	</ol>
	</li>
	<li>Analysis of flavor by GC-MS
	<ol>
		<li>Refer to the Table of Materials for details of the instrument used.</li>
		<li>Use Helium (purity &#62; 99.995%) as the carrier gas, and set the flow rate to 0.9 mL/min.</li>
		<li>Set the column temperature to 40 &#176;C for 3 min initially, and then increase to 90 &#176;C at a rate of 5 &#176;C/min. Subsequently, increase to 230 &#176;C at a rate of 10 &#176;C/min.</li>
		<li>Run MS in electron ionization (EI)+ mode, and set electron energy as 70 eV. Set scan range from 30 m/z to 500 m/z. Set emission current to 80 &#956;A. Use interface and source temperatures of 250 and 200 &#176;C, respectively.</li>
		<li>Identify the volatile flavor compounds by NIST2005 and Willey 7 standard libraries. Semi-quantify the retention index (RI) of volatile compounds using an internal standard (2,4,6-trimethylpyridine).</li>
		<li>Calculate the concentration of volatile flavor compounds by comparing the peak area of each flavor compound with that of the internal standard. Express the results as &#956;g/kg.</li>
	</ol>
	</li>
</ol>
5. Texture profile analysis
NOTE: Analyze texture profile by following a previous study20.
<ol>
	<li>Perform texture profile analysis (TPA) with a texture analyzer equipped with a cylindrical probe (P/36R).</li>
	<li>Apply two consecutive cycles. Deformation degree of TPA is 50% and the trigger force is 5 x g.</li>
	<li>The speed of cylindrical probe for pre-, in- and pro-test, are 2, 1 and 5 mm/s, respectively. Calculate the texture parameters by its inbuilt software.</li>
</ol>
6. Color measurement
NOTE: Measure color according to the method of Czerner et al. with some modifications21.
<ol>
	<li>Measure samples by using colorimeter. Record the lightness value (L*), greenness/redness value (a*) and blueness/yellowness value (b*) of the samples.</li>
	<li>Measure five replicates for each sample.</li>
</ol>
7. Sensory evaluation
<ol>
	<li>Conduct sensory analysis of samples by at least 20 trained panelists (23 panelists as an example, 12 man and 11 women, ages 20-40) using a previously method with some modifications20.</li>
	<li>Score the samples from 0 to 10 for taste, flavor, color and texture. Score 0 represents &#34;dislike extremely&#34;&#160;and score 10 represents for &#34;like extremely&#34;. 
	NOTE: Scoring criteria are shown in Table 1. The overall score is composed of a sum of different contributors (30% taste score + 30% flavor score + 20% color score + 20% texture score). An overall score of 6 is viewed as the borderline of acceptable quality.</li>
</ol>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Quality parameter</td>
			<td>Description</td>
			<td>Score</td>
		</tr>
		<tr>
			<td rowspan="4">Taste</td>
			<td>Acceptable sweet and salty; harmonious taste; wine taste; not astringent</td>
			<td>8-10</td>
		</tr>
		<tr>
			<td>Too heavy or light (sweet or salty); harmonious taste; wine taste; not astringent</td>
			<td>6-8</td>
		</tr>
		<tr>
			<td>Too heavy or light (sweet or salty); light wine- and after- taste; astringent</td>
			<td>3-6</td>
		</tr>
		<tr>
			<td>Too light wine- and after- taste; obviously astringent</td>
			<td>0-3</td>
		</tr>
		<tr>
			<td rowspan="4">Flavor</td>
			<td>Mellow wine flavor; rich fermentation aroma; no weird smell</td>
			<td>8-10</td>
		</tr>
		<tr>
			<td>Mellow wine flavor; light fermentation aroma; no weird smell</td>
			<td>6-8</td>
		</tr>
		<tr>
			<td>Light wine flavor; light fermentation aroma; light weird smell</td>
			<td>3-6</td>
		</tr>
		<tr>
			<td>No wine aroma; light fermentation aroma; obviously weird smell</td>
			<td>0-3</td>
		</tr>
		<tr>
			<td rowspan="4">Appearance</td>
			<td>Russet color; glossy appearance; bright surface</td>
			<td>8-10</td>
		</tr>
		<tr>
			<td>Russet color; bright surface</td>
			<td>6-8</td>
		</tr>
		<tr>
			<td>Yellow color; nonuniform</td>
			<td>3-6</td>
		</tr>
		<tr>
			<td>Pale color; rough surface</td>
			<td>0-3</td>
		</tr>
		<tr>
			<td rowspan="4">Texture</td>
			<td>Acceptable chewiness</td>
			<td>8-10</td>
		</tr>
		<tr>
			<td>Hard</td>
			<td>6-8</td>
		</tr>
		<tr>
			<td>Light springiness</td>
			<td>3-6</td>
		</tr>
		<tr>
			<td>Soft texture; coarse mouth-feel</td>
			<td>0-3</td>
		</tr>
	</tbody>
</table>
Table 1: Scoring criteria for sensory quality of fermented wine-aroma sturgeon meat product.

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The authors have nothing to disclose.

In this study, a new technique for the production of high-quality fermented fish product with wine aroma and tests for sensory properties are provided. The key processes of this technique are marinating, drying, fermentation and sterilization. In the marinating process, the salt concentration, the temperature and the time all affect the textural properties of the fish. The hardness and chewiness of the product increase gradually with the increase of salt concentration (0-12%, w/v) and the prolongation of marinating time ...

Current commercial marinated fish product in China has the problem of heavy salty taste, insufficient wine aroma, poor elasticity and pale color, which decreases the acceptability to consumers. Therefore, a new technique for a high-quality fish meat product with wine aroma needs to be created and optimized.
In recent years, application of modern fermentation techniques in meat and fish has attracted attention from more and more researchers1,2,3,4. By inoculation of starter cultures into meat and fish, food safety has been enhanced, the processing time has been shortened; and the product sensory properties have been modified. Saithong et al.5 isolated lactobacillus bacteria (LAB) from natural plaa-som and used this LAB as a starter culture, which induced high acidity and suppressed pathogenic bacteria. Zeng et al.6 reported that inoculation with the autochthonous starter cultures reduced fermentation time and improved the sensory properties of samples. Casaburi et al.7 claimed that the use of microbial starter cultures influence the development of aroma in fermented meats. In these starter cultures, S. cerevisiae could produce wine aroma by alcoholic fermentation and could also give the product other improved organoleptic qualities. Therefore, S. cerevisiae is a suitable starter culture for wine-aroma products8,9,10 and wine-aroma fish product could be made by S. cerevisiae.
In the process of making wine-aroma fish product, the texture of meat and fish could be affected by salt content, water content, pH, protein denaturation, etc. Therefore, marinating, drying, fermentation and sterilization could all influence the texture characteristics. The formation of flavor and taste is complicated and is mainly affected by marinating and fermentation, because it is highly related to hydrolysis of carbohydrates, proteins and lipids, and mild lipid oxidation11,12. It could also be affected by addition of spices13. For development of color, Maillard reaction occurs which is involved in the process of fermentation and sterilization10.
This article could provide technical support for the industrialization of fermented fish product with wine aroma, which is of great significance to the development of the fish processing industry. This technique could improve taste of product by increased proteolysis (more free amino acids and TCA-soluble peptides), modify flavor mainly by alcohols (ethanol, 1-octen-3-ol, 2-methyl-1-propanol and 3-methyl-1-butanol), esters (ethyl acetate) and aldehydes (nonanal, 3-methylbutanal and benzaldehyde), increase mouthfeel by higher hardness, springiness, gumminess and chewiness, and give more attractive russet color by and a bright surface14. It also gives consumers convenience because the product can be stored at room temperature. As described by other previous studies15,16,17, fermentation with S. cerevisiae has also been proved to significantly improve organoleptic qualities in other meat or fish products.
It is worth noting that the introduced protocol could also be applied in other species of fish, such as grass carp, silver carp, black carp, bighead carp, cod, salmon, etc. For high quality of fish products, fish without processing should be used, such as fresh fish, fish in ice or frozen fish stored for less than 1 year. Besides, since mild lipid oxidation could enhance flavor while extensive lipid oxidation brings unpleasant flavor, fish with less fat is preferred or skimmed fish is recommended.

<table><tbody><tr><td>2,4,6-trimethylpyridine</td><td>Tokyo Chemical Industry Co., Ltd.</td><td>Purity 98%</td><td></td></tr><tr><td>Colorimeter</td><td>Hunterlab</td><td>UltraScan Pro1166</td><td></td></tr><tr><td>DB-WAX column</td><td>Agilent</td><td>30 m &amp;times; 0.25 mm &amp;times; 0.25&amp;mu;m</td><td></td></tr><tr><td>Digital pH meter</td><td>Mettler toledo Instrument (Shanghai) Co., Ltd.</td><td>DELTA-320</td><td></td></tr><tr><td>Drying oven</td><td>Shanghai Yiheng Scientific Instrument Co., Ltd.</td><td>DHG-9070A</td><td></td></tr><tr><td>Frozen sturgeon</td><td>Huada Marine Industry Group Co., Ltd</td><td>-</td><td></td></tr><tr><td>Gas chromatograph-mass spectrometer</td><td>Thermo Fisher Scientific</td><td>TSQ Quantum XLS</td><td></td></tr><tr><td>Humidities incubator</td><td>Shanghai Yiheng Scientific Instrument Co., Ltd.</td><td>LHS-250HC-II</td><td></td></tr><tr><td>&lt;em&gt;Saccharomyces cerevisiae&lt;/em&gt;</td><td>Angel Yeast Co., Ltd</td><td>-</td><td></td></tr><tr><td>Spices</td><td>Auchan Supermarket</td><td>-</td><td></td></tr><tr><td>Sterilization pot</td><td>Longqiang Machinery Technology Co., Ltd.</td><td>RHS-03-700</td><td></td></tr><tr><td>Supelco</td><td>Sigma</td><td>65&amp;mu;m, PDMS/DVB</td><td></td></tr><tr><td>Texture analyzer</td><td>Stable Micro Systems, Ltd.</td><td>TA-XT2i</td><td></td></tr><tr><td>Vacuum package machine</td><td>Quanzhou Yiminxin Electromechanical Co., Ltd.</td><td>YMX-958-10L</td><td></td></tr></tbody></table>

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 suitable salt concentration, marinating time and temperature make the product&#8217;s textural quality better. The best marinating conditions were as follows: the salt concentration of 8% in the marinating solution; marinating time of 2 h; and marinating temperature of 10 &#176;C. See Figure 1.
In drying process, the final moisture content and drying temperature could influence the texture and sensory quality. The best drying conditions were as follows: moistu...

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Preparation of High-Quality Fermented Fish Product

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 ...

preparation-of-high-quality-fermented-fish-product

Universidad San Sebastian

Research

JoVE Journal

Biochemistry

10.3K Views.  Jiangnan University. The goal of the protocol is to provide an industrialized fish fermentation technique based on inoculation of Saccharomyces cerevisiae.

Video: Preparation of High-Quality Fermented Fish Product

Laboratory Estimation of Net Trophic Transfer Efficiencies of PCB Congeners to Lake Trout (Salvelinus namaycush) from Its Prey

A technique for laboratory estimation of net trophic transfer efficiency (&#947;) of polychlorinated biphenyl (PCB) congeners to piscivorous fish from their prey is described herein. During a 135-day laboratory experiment, we fed bloater (Coregonus hoyi) that had been caught in Lake Michigan to lake trout (Salvelinus namaycush) kept in eight laboratory tanks. Bloater is a natural prey for lake trout. In four of the tanks, a relatively high flow rate was used to ensure relatively high activity by the lake trout, whereas a low flow rate was used in the other four tanks, allowing for low lake trout activity. On a tank-by-tank basis, the amount of food eaten by the lake trout on each day of the experiment was recorded. Each lake trout was weighed at the start and end of the experiment. Four to nine lake trout from each of the eight tanks were sacrificed at the start of the experiment, and all 10 lake trout remaining in each of the tanks were euthanized at the end of the experiment. We determined concentrations of 75 PCB congeners in the lake trout at the start of the experiment, in the lake trout at the end of the experiment, and in bloaters fed to the lake trout during the experiment. Based on these measurements, &#947; was calculated for each of 75 PCB congeners in each of the eight tanks. Mean &#947; was calculated for each of the 75 PCB congeners for both active and inactive lake trout. Because the experiment was replicated in eight tanks, the standard error about mean &#947; could be estimated. Results from this type of experiment are useful in risk assessment models to predict future risk to humans and wildlife eating contaminated fish under various scenarios of environmental contamination.

Laboratory Estimation of Net Trophic Transfer Efficiencies of PCB Congeners to Lake Trout Salvelinus namaycush from Its Prey

A technique for laboratory estimation of net trophic transfer efficiency of polychlorinated biphenyl (PCB) congeners to piscivorous fish from their prey is presented. To maximize applicability of the laboratory results to the field, the piscivorous fish should be fed prey fish that are typically eaten in the field.

A technique for laboratory estimation of net trophic transfer efficiency (&#947;) of polychlorinated biphenyl (PCB) congeners to piscivorous fish from ...

Environment

The Complete and Updated "Rotifer Polyculture Method" for Rearing First Feeding Zebrafish

The zebrafish (Danio rerio) is a model organism of increasing importance in many fields of science. One of the most demanding technical aspects of culture of this species in the laboratory is rearing first-feeding larvae to the juvenile stage with high rates of growth and survival. The central management challenge of this developmental period revolves around delivering highly nutritious feed items to the fish on a nearly continuous basis without compromising water quality. Because larval zebrafish are well-adapted to feed on small zooplankton in the water column, live prey items such as brachionid rotifers, Artemia, and Paramecium are widely recognized as the feeds of choice, at least until the fish reach the juvenile stage and are able to efficiently feed on processed diets. This protocol describes a method whereby newly hatched zebrafish larvae are cultured together with live saltwater rotifers (Brachionus plicatilis) in the same system. This polyculture approach provides fish with an "on-demand", nutrient-rich live food source without producing chemical waste at levels that would otherwise limit performance. Importantly, because the system harnesses both the natural high productivity of the rotifers and the behavioral preferences of the fish, the labor involved with maintenance is low. The following protocol details an updated, step-by-step procedure that incorporates rotifer production (scalable to any desired level) for use in a polyculture of zebrafish larvae and rotifers that promotes maximal performance during the first 5 days of exogenous feeding.

Larval zebrafish are adapted to feed on zooplankton. It is possible to capitalize on this natural feature in the laboratory by growing first feeding fish together in the same system with live saltwater rotifers. This "polyculture" strategy promotes high growth and survival with minimal labor and disturbance to the larvae.

The zebrafish (Danio rerio) is a model organism of increasing importance in many fields of science.  One of the most demanding technical aspects of ...

Developmental Biology

A 3-dimensional (3D)-printed Template for High Throughput Zebrafish Embryo Arraying

The zebrafish is a globally recognized fresh water organism frequently used in developmental biology, environmental toxicology, and human disease related research fields. Thanks to its unique features, including large fecundity, embryo translucency, rapid and simultaneous development, etc., zebrafish embryos are often used for large scale toxicity assessment of chemicals and drug/compound screening. A typical screening procedure involves adult zebrafish spawning, embryos selection, and arraying the embryos into multi-well plates. From there, embryos are subjected to exposure and the toxicity of chemical, or the effectiveness of the drugs/compounds can be evaluated relatively quickly based on phenotypic observations. Among these processes, embryos arraying is one of the most time-consuming and labor-intensive steps that limits the throughput level. In this protocol, we present an innovative approach that makes use of a 3D-printed arraying template coupled with vacuum manipulation to speed up this laborious step. The protocol herein describes the overall design of the arraying template, a detailed experimental setup and step-by-step procedure, followed by representative results. When implemented, this approach should prove beneficial in a variety of research applications using zebrafish embryos as testing subjects.

A 3-dimensional 3D-printed Template for High Throughput Zebrafish Embryo Arraying

Here, we present a protocol to design and fabricate a zebrafish embryo arraying template, followed by a detailed procedure on the use of such template for high throughput zebrafish embryo arraying into a 96-well plate.

The zebrafish is a globally recognized fresh water organism frequently used in developmental biology, environmental toxicology, and human disease related ...

Optimizing GF Zebrafish Models: Generation, Maintenance, and Microbial Studies

Generation, Maintenance, and Identification of Germ-Free Zebrafish Models from Larvae to Juvenile Stages

Zebrafish serve as valuable models for research on growth, immunity, and gut microbiota due to their genomic similarities with mammals, transparent embryos developed in a relatively clean chorion environment, and extremely rapid development of larvae compared to rodent models. Germ-free (GF) zebrafish (Danio rerio) are crucial for evaluating pollutant toxicity and establishing human-like disease models related to microbial functions. In comparison to conventionally raised (CR) models (fish in common husbandry), GF zebrafish allow for more accurate manipulation of the host microbiota, aiding in determining the causal relationship between microorganisms and hosts. Consequently, they play a critical role in advancing our understanding of these relationships. However, GF zebrafish models are typically generated and researched during the early life stages (from embryos to larvae) due to limitations in immune function and nutrient absorption. This study optimizes the generation, maintenance, and identification of early GF zebrafish models without feeding and with long-term feeding using GF food (such as Artemia sp., brine shrimp). Throughout the process, daily sampling and culture were performed and identified through multiple detections, including plates and 16S rRNA sequencing. The aseptic rate, survival, and developmental indexes of GF zebrafish were recorded to ensure the quality and quantity of the generated models. Importantly, this study provides details on bacterial isolation and infection techniques for GF fish, enabling the efficient creation of GF fish models from larvae to juvenile stages with GF food support. By applying these procedures in biomedical research, scientists can better understand the relationships between intestinal bacterial functions and host health.

Author Spotlight: Establishing Germ-Free Zebrafish for Microbiota-Host Relationship Studies

This protocol outlines the primary steps for obtaining germ-free (GF) fish embryos and maintaining them from larvae until the juvenile stage, including sampling and detecting their sterile status. The use of GF models with infection is important for understanding the role of microbes in host health.

Zebrafish serve as valuable models for research on growth, immunity, and gut microbiota due to their genomic similarities with mammals, transparent ...

Preparation of a High-quality Primary Cell Culture from Fish Pituitaries

Primary cell culture is a powerful tool commonly used by scientists to study cellular properties and mechanisms of isolated cells in a controlled environment. Despite vast differences in the physiology between mammals and fish, primary cell culture protocols from fish are often based on mammalian culture conditions, often with only minor modifications. The environmental differences affect not only body temperature, but also blood serum parameters such as osmolality, pH, and pH buffer capacity. As cell culture media and similar working solutions are meant to mimic characteristics of the extracellular fluid and/or blood serum to which a cell is adapted, it is crucial that these parameters are adjusted specifically to the animal in question.
The current protocol describes optimized primary culture conditions for medaka (Oryzias latipes). The protocol provides detailed steps on how to isolate and maintain healthy dissociated pituitary cells for more than one week and includes the following steps: 1. the adjustment of the osmolality to the values found in medaka blood plasma, 2. the adjustment of the incubation temperature to normal medaka temperature (here in the aquarium facility), and 3. the adjustment of the pH and bicarbonate buffer to values comparable to other fish species living at similar temperatures. The results presented using the described protocol promote physiologically meaningful results for medaka and can be used as a reference guide by scientists making primary cell cultures from other non-mammalian species.

Here we describe a protocol to prepare and maintain primary pituitary cell cultures from medaka (Oryzias latipes). The optimized conditions in this protocol take important parameters such as temperature, osmolality, and pH into consideration by mimicking the physiological conditions of the fish, thereby enabling physiologically more meaningful results.

Primary cell culture is a powerful tool commonly used by scientists to study cellular properties and mechanisms of isolated cells in a controlled ...

Neuroscience

Light-Controlled Fermentations for Microbial Chemical and Protein Production

Microbial cell factories offer a sustainable alternative for producing chemicals and recombinant proteins from renewable feedstocks. However, overburdening a microorganism with genetic modifications can reduce host fitness and productivity. This problem can be overcome by using dynamic control: inducible expression of enzymes and pathways, typically using chemical- or nutrient-based additives, to balance cellular growth and production. Optogenetics offers a non-invasive, highly tunable, and reversible method of dynamically regulating gene expression. Here, we describe how to set up light-controlled fermentations of engineered Escherichia coli and Saccharomyces cerevisiae for the production of chemicals or recombinant proteins. We discuss how to apply light at selected times and dosages to decouple microbial growth and production for improved fermentation control and productivity, as well as the key optimization considerations for best results. Additionally, we describe how to implement light controls for lab-scale bioreactor experiments. These protocols facilitate the adoption of optogenetic controls in engineered microorganisms for improved fermentation performance.

Optogenetic control of microbial metabolism offers flexible dynamic control over fermentation processes. The protocol here shows how to set up blue light-regulated fermentations for chemical and protein production at different volumetric scales.

Microbial cell factories offer a sustainable alternative for producing chemicals and recombinant proteins from renewable feedstocks. However, ...

Bioengineering

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 ...

Biology

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 ...

Single-Cell Suspension Preparation from Nile Tilapia Intestine for Single-Cell Sequencing

Nile tilapia is one of the most commonly cultured freshwater fish species worldwide and is a widely used research model for aquaculture fish studies. The preparation of high-quality single-cell suspensions is essential for single-cell level studies such as single-cell RNA or genome sequencing. However, there is no ready-to-use protocol for aquaculture fish species, particularly for the intestine of tilapia. The effective dissociation enzymes vary depending on the tissue type. Therefore, optimizing the tissue dissociation protocol by selecting the appropriate enzyme or enzyme combination to obtain enough viable cells with minimum damage is essential. This study illustrates an optimized protocol to prepare a high-quality single-cell suspension from Nile tilapia intestine with an enzyme combination of collagenase/dispase. This combination is highly effective for dissociation with the utilization of bovine serum albumin and DNase to reduce cell aggregation after digestion. The cell output satisfies the requirements for single-cell sequencing, with 90% cell viability and a high cell concentration. This protocol can also be modified to prepare a single-cell suspension from the intestines of other fish species. This research provides an efficient reference protocol and reduces the need for additional trials in the preparation of single-cell suspensions for aquaculture fish species.

Here, we demonstrate the preparation of a high-quality single-cell suspension of tilapia intestine for single-cell sequencing.

Nile tilapia is one of the most commonly cultured freshwater fish species worldwide and is a widely used research model for aquaculture fish studies. The ...

Optimizing Spray-Dried Probiotic Product Quality Through Process Development

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

Probiotics and prebiotics are of great interest to the food and pharmaceutical industries due to their health benefits. Probiotics are live bacteria that can confer beneficial effects on human and animal wellbeing, while prebiotics are types of nutrients that feed the beneficial gut bacteria. Powder probiotics have gained popularity due to the ease and practicality of their ingestion and incorporation into the diet as a food supplement. However, the drying process interferes with cell viability since high temperatures inactivate probiotic bacteria. In this context, this study aimed to present all the steps involved in the production and physicochemical characterization of a spray-dried probiotic and evaluate the influence of the protectants (simulated skim milk and inulin:maltodextrin association) and drying temperatures in increasing the powder yield and cell viability. The results showed that the simulated skim milk promoted higher probiotic viability at 80 &#176;C. With this protectant, the probiotic viability, moisture content, and water activity (Aw) reduce as long as the inlet temperature increases. The probiotics&#39; viability decreases conversely with the drying temperature.&#160;At temperatures close to 120 &#176;C, the dried probiotic showed viability around 90%, a moisture content of 4.6% w/w, and an Aw of 0.26; values adequate to guarantee product stability. In this context, spray-drying temperatures above 120 &#176;C are required to ensure the microbial cells&#39; viability and shelf-life in the powdered preparation and survival during food processing and storage.

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

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

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