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.

<ol>
	<li>Riebroy, S., Benjakul, S., Visessanguan, W., Tanaka, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+during+fermentation+and+properties+of+som-fug+produced+from+different+marine+fish.">Changes during fermentation and properties of som-fug produced from different marine fish.</a> Journal of Food Processing and Preservation. 31 (6), 751-770 (2007).</li><li>Xu, Y., Xia, W., Yang, F., Jinmoon, K., Nie, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+fermentation+temperature+on+the+microbial+and+physicochemical+properties+of+silver+carp+sausages+inoculated+with+Pediococcus+pentosaceus.">Effect of fermentation temperature on the microbial and physicochemical properties of silver carp sausages inoculated with Pediococcus pentosaceus.</a> Food Chemistry. 118 (3), 512-518 (2010).</li><li>Cano-Garcia, L., Flores, M., Belloch, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+characterization+and+aromatic+potential+of+Debaryomyces+hansenii+strains+isolated+from+naturally+fermented+sausages.">Molecular characterization and aromatic potential of Debaryomyces hansenii strains isolated from naturally fermented sausages.</a> Food Research International. 52 (1), 42-49 (2013).</li><li>Chen, Q., Liu, Q., Sun, Q., Kong, B., Xiong, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flavour+formation+from+hydrolysis+of+pork+sarcoplasmic+protein+extract+by+a+unique+LAB+culture+isolated+from+Harbin+dry+sausage.">Flavour formation from hydrolysis of pork sarcoplasmic protein extract by a unique LAB culture isolated from Harbin dry sausage.</a> Meat Science. 100, 110-117 (2015).</li><li>Saithong, P., Panthavee, W., Boonyaratanakornkit, M., Sikkhamondhol, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+a+starter+culture+of+lactic+acid+bacteria+in+plaa-som,+a+Thai+fermented+fish.">Use of a starter culture of lactic acid bacteria in plaa-som, a Thai fermented fish.</a> Journal of Bioscience &amp; Bioengineering. 110 (5), 553-557 (2010).</li><li>Zeng, X., Xia, W., Jiang, Q., Guan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+and+Sensory+Characteristics+of+Whole+Carp+Inoculated+With+Autochthonous+Starter+Cultures.">Biochemical and Sensory Characteristics of Whole Carp Inoculated With Autochthonous Starter Cultures.</a> Journal of Aquatic Food Product Technology. 24 (1), 52-67 (2015).</li><li>Casaburi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+and+sensory+characteristics+of+traditional+fermented+sausages+of+Vallo+di+Diano+(Southern+Italy)+as+affected+by+the+use+of+starter+cultures.">Biochemical and sensory characteristics of traditional fermented sausages of Vallo di Diano (Southern Italy) as affected by the use of starter cultures.</a> Meat Science. 76 (2), 295-307 (2007).</li><li>Gao, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+autochthonous+starter+cultures+on+the+volatile+flavour+compounds+of+Chinese+traditional+fermented+fish+(Suan+yu).">Effect of autochthonous starter cultures on the volatile flavour compounds of Chinese traditional fermented fish (Suan yu).</a> International Journal of Food Science and Technology. 51 (7), 1630-1637 (2016).</li><li>Zeng, X. F., Xia, W. S., Jiang, Q. X., Xu, Y. S., Fan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+Mixed+Starter+Cultures+to+Flavor+Profile+of+Suanyu+-+A+Traditional+Chinese+Low-Salt+Fermented+Whole+Fish.">Contribution of Mixed Starter Cultures to Flavor Profile of Suanyu - A Traditional Chinese Low-Salt Fermented Whole Fish.</a> Journal of Food Processing and Preservation. 41 (5), 15 (2017).</li><li>Anihouvi, V. B., Sakyi-Dawson, E., Ayernor, G. S., Hounhouigan, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbiological+changes+in+naturally+fermented+cassava+fish+(Pseudotolithus+sp.)+for+lanhouin+production.">Microbiological changes in naturally fermented cassava fish (Pseudotolithus sp.) for lanhouin production.</a> International Journal of Food Microbiology. 116 (2), 287-291 (2007).</li><li>Corral, S., Salvador, A., Flores, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salt+reduction+in+slow+fermented+sausages+affects+the+generation+of+aroma+active+compounds.">Salt reduction in slow fermented sausages affects the generation of aroma active compounds.</a> Meat Science. 93 (3), 776-785 (2013).</li><li>Wang, W., Xia, W., Pei, G., Xu, Y., Jiang, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteolysis+during+fermentation+of+Suanyu+as+a+traditional+fermented+fish+product+of+China.">Proteolysis during fermentation of Suanyu as a traditional fermented fish product of China.</a> International Journal of Food Properties. 20 (90), 29-38 (2017).</li><li>Coventry, M. J., Hickey, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+spices+and+manganese+on+meat+starter+culture+activity.">The effect of spices and manganese on meat starter culture activity.</a> Meat Science. 33 (3), 391-399 (1993).</li><li>Zhu, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+study+on+quality+characteristics+of+pickled+and+fermented+sturgeon+(Acipenser+sinensis)+meat+in+retort+cooking.">Comparative study on quality characteristics of pickled and fermented sturgeon (Acipenser sinensis) meat in retort cooking.</a> International Journal of Food Science &amp; Technology. , (2019).</li><li>Mendonca, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+the+yeast+flora+in+artisanal+country+style+and+industrial;dry+cured+sausage+(yeast+in+fermented+sausage).">Dynamics of the yeast flora in artisanal country style and industrial;dry cured sausage (yeast in fermented sausage).</a> Food Control. 29 (1), 143-148 (2013).</li><li>Zeng, X. F., Xia, W. S., Jiang, Q. X., Yang, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+autochthonous+starter+cultures+on+microbiological+and+physico-chemical+characteristics+of+Suan+yu,+a+traditional+Chinese+low+salt+fermented+fish.">Effect of autochthonous starter cultures on microbiological and physico-chemical characteristics of Suan yu, a traditional Chinese low salt fermented fish.</a> Food Control. 33 (2), 344-351 (2013).</li><li>Gao, P., Jiang, Q., Xu, Y., Xia, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Esterase+activities+of+autochthonous+starter+cultures+to+increase+volatile+flavor+compounds+in+Chinese+traditional+fermented+fish+(Suan+yu).">Esterase activities of autochthonous starter cultures to increase volatile flavor compounds in Chinese traditional fermented fish (Suan yu).</a> International Journal of Food Properties. 20 (1), 663-672 (2017).</li><li>Wahyuni, S., Holilah, , Asranudin, , Noviyanti, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimation+of+shelf+life+of+wikau+maombo+brownies+cake+using+Accelerated+Shelf+Life+Testing+(ASLT)+method+with+Arrhenius+model.">Estimation of shelf life of wikau maombo brownies cake using Accelerated Shelf Life Testing (ASLT) method with Arrhenius model.</a> IOP Conference Series Earth and Environmental Science. 122 (1), 12-22 (2018).</li><li>Zeng, X. F., Xia, W. S., Jiang, Q. X., Yang, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+and+microbial+properties+of+Chinese+traditional+low-salt+fermented+whole+fish+product+Suan+yu.">Chemical and microbial properties of Chinese traditional low-salt fermented whole fish product Suan yu.</a> Food Control. 30 (2), 590-595 (2013).</li><li>Yu, D., Jiang, Q., Xu, Y., Xia, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+shelf+life+extension+of+refrigerated+grass+carp+(Ctenopharyngodon+idellus)+fillets+by+chitosan+coating+combined+with+glycerol+monolaurate.">The shelf life extension of refrigerated grass carp (Ctenopharyngodon idellus) fillets by chitosan coating combined with glycerol monolaurate.</a> International Journal of Biological Macromolecules. 101, 448-454 (2017).</li><li>Czerner, M., Tomas, M. C., Yeannes, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ripening+of+salted+anchovy+(Engraulis+anchoita):+development+of+lipid+oxidation,+colour+and+other+sensorial+characteristics.">Ripening of salted anchovy (Engraulis anchoita): development of lipid oxidation, colour and other sensorial characteristics.</a> Journal of the Science of Food and Agriculture. 91 (4), 609-615 (2011).</li></ol>

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 &#215; 0.25 mm &#215; 0.25&#956;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>Saccharomyces cerevisiae</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&#956;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

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JoVE Journal

Biochemistry

10.1K 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

Preparation of Homogeneous MALDI Samples for Quantitative Applications

This protocol demonstrates a simple sample preparation to reduce spatial heterogeneity in ion signals during matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The heterogeneity of ion signals is a severe problem in MALDI, which results in poor data reproducibility and makes MALDI unsuitable for quantitative analysis. By regulating sample plate temperature during sample preparation, thermal-induced hydrodynamic flows inside droplets of sample solution are able to reduce the heterogeneity problem. A room-temperature sample preparation chamber equipped with a temperature-regulated copper base block that holds MALDI sample plates facilitates precise control of the sample drying condition. After drying of sample droplets, the temperature of sample plates is returned to room temperature and removed from the chamber for subsequent mass spectrometric analysis. The areas of samples are examined with MALDI-imaging mass spectrometry to obtain the spatial distribution of all components in the sample. In comparison with the conventional dried-droplet method that prepares samples under ambient conditions without temperature control, the samples prepared with the method demonstrated herein show significantly better spatial distribution and signal intensity. According to observations using carbohydrate and peptide samples, decreasing substrate temperature while maintaining the surroundings at ambient temperature during the drying process can effectively reduce the heterogeneity of ion signals. This method is generally applicable to various combinations of samples and matrices.

A protocol for reducing spatial heterogeneities of ion signals in MALDI mass spectrometry by regulating substrate temperature during sample drying processes is demonstrated.

This protocol demonstrates a simple sample preparation to reduce spatial heterogeneity in ion signals during matrix-assisted laser desorption/ionization ...

A Filtration-based Method of Preparing High-quality Nuclei from Cross-linked Skeletal Muscle for Chromatin Immunoprecipitation

Chromatin immunoprecipitation (ChIP) is a powerful method to determine protein binding to chromatin DNA. Fiber-rich skeletal muscle, however, has been a challenge for ChIP due to technical difficulty in isolation of high-quality nuclei with minimal contamination of myofibrils. Previous protocols have attempted to purify nuclei before cross-linking, which incurs the risk of altered DNA-protein interaction during the prolonged nuclei preparation process. In the current protocol, we first cross-linked the skeletal muscle tissue collected from mice, and the tissues were minced and sonicated. Since we found that ultracentrifugation was not able to separate nuclei from myofibrils using cross-linked muscle tissue, we devised a sequential filtration procedure to obtain high-quality nuclei devoid of significant myofibril contamination. We subsequently prepared chromatin by using an ultrasonicator, and ChIP assays with anti-BMAL1 antibody revealed robust circadian binding pattern of BMAL1 to target gene promoters. This filtration protocol constitutes an easily applicable method to isolate high-quality nuclei from cross-linked skeletal muscle tissue, allowing consistent sample processing for circadian and other time-sensitive studies. In combination with next-generation sequencing (NGS), our method can be deployed for various mechanistic and genomic studies focusing on skeletal muscle function.

We present a filtration-based protocol to isolate high-quality nuclei from cross-linked mouse skeletal muscle wherein we removed the need for ultracentrifugation, making it easily applicable. We show that chromatin prepared from the nuclei is suitable for chromatin immunoprecipitation and likely chromatin immunoprecipitation sequencing studies.

Chromatin immunoprecipitation (ChIP) is a powerful method to determine protein binding to chromatin DNA. Fiber-rich skeletal muscle, however, has been a ...

Biosynthesis of a Flavonol from a Flavanone by Establishing a One-pot Bienzymatic Cascade

Flavonols are a major subclass of flavonoids with a variety of biological and pharmacological activities. Here, we provide a method for the in vitro enzymatic synthesis of a flavonol. In this method, Atf3h and Atfls1, two key genes in the biosynthetic pathway of the flavonols, are cloned and overexpressed in Escherichia coli. The recombinant enzymes are purified via an affinity column and then a bienzymatic cascade is established in a specific synthetic buffer. Two flavonols are synthesized in this system as examples and determined by TLC and HPLC/LC/MS analyses. The method displays obvious advantages in the derivation of flavonols over other approaches. It is time- and labor-saving and highly cost-effective. The reaction is easy to be accurately controlled and thus scaled up for mass production. The target product can be purified easily due to the simple components in the system. However, this system is usually restricted to the production of a flavonol from a flavanone.

The derivation of a flavonol is crucial for its application in healthcare and the food industry. Here, we provide a detailed protocol for the biosynthesis of a flavonol from a flavanone and discuss the crucial steps and its advantages over other approaches.

Flavonols are a major subclass of flavonoids with a variety of biological and pharmacological activities. Here, we provide a method for the in vitro ...

Obtaining High-Quality Transcriptome Data from Cereal Seeds by a Modified Method for Gene Expression Profiling

The characterization of gene expression is dependent on RNA quality. In germinating, developing and mature cereal seeds, the extraction of high-quality RNA is often hindered by high starch and sugar content. These compounds can reduce both the yield and the quality of the extracted total RNA. The deterioration in quantity and quality of total RNA can subsequently have a significant impact on the downstream transcriptomic analyses, which may not accurately reflect the spatial and/or temporal variation in the gene expression profile of the samples being tested. In this protocol, we describe an optimized method for extraction of total RNA with sufficient quantity and quality to be used for whole transcriptome analysis of cereal grains. The described method is suitable for several downstream applications used for transcriptomic profiling of developing, germinating, and mature cereal seeds. The method of transcriptome profiling using a microarray platform is shown. This method is specifically designed for gene expression profiling of cereals with described genome sequences. The detailed procedure from microarray handling to final quality control is described. This includes cDNA synthesis, cRNA labelling, microarray hybridization, slide scanning, feature extraction, and data quality validation. The data generated by this method can be used to characterize the transcriptome of cereals during germination, in various stages of grain development, or at different biotic or abiotic stress conditions. The results presented here exemplify high-quality transcriptome data amenable for downstream bioinformatics analyses, such as the determination of differentially expressed genes (DEGs), characterisation of gene regulatory networks, and conducting transcriptome-wide association study (TWAS).

A method for transcriptome profiling of cereals is presented. The microarray-based gene expression profiling starts with the isolation of high-quality total RNA from cereal grains and continues with the generation of cDNA. After cRNA labelling and microarray hybridization, recommendations are given for signal detection and quality control.

The characterization of gene expression is dependent on RNA quality. In germinating, developing and mature cereal seeds, the extraction of high-quality ...

Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk population methods (Northern blot, PCR, and RNAseq) that measure mRNA levels lack the ability to provide information on cell-to-cell variation in responses. Precise single cell and allelic visualization and quantification is possible via single molecule RNA fluorescence in situ hybridization (smFISH). RNA-FISH is performed by hybridizing target RNAs with labeled oligonucleotide probes. These can be imaged in medium/high throughput modalities, and, through image analysis pipelines, provide quantitative data on both mature and nascent RNAs, all at the single cell level. The fixation, permeabilization, hybridization and imaging steps have been optimized in the lab over many years using the model system described herein, which results in successful and robust single cell analysis of smFISH labeling. The main goal with sample preparation and processing is to produce high quality images characterized by a high signal-to-noise ratio to reduce false positives and provide data that are more accurate. Here, we present a protocol describing the pipeline from sample preparation to data analysis in conjunction with suggestions and optimization steps to tailor to specific samples.&#160;

Single molecule RNA fluorescence in situ hybridization (smFISH) is a method to accurately quantify levels and localization of specific RNAs at the single cell level. Here, we report our validated lab protocols for wet-bench processing, imaging and image analysis for single cell quantification of specific RNAs.

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk ...

Nano-Differential Scanning Fluorimetry for Screening in Fragment-based Lead Discovery

Thermal shift assays (TSAs) examine how the melting temperature (Tm) of a target protein changes in response to changes in its environment (e.g., buffer composition). The utility of TSA, and specifically of nano-Differential Scanning Fluorimetry (nano-DSF), has been established over the years, both for finding conditions that help stabilize a specific protein and for looking at ligand binding by monitoring changes in the apparent Tm. This paper presents an efficient screening of the Diamond-SGC-iNEXT Poised (DSi-Poised) fragment library (768 compounds) by the use of nano-DSF, monitoring Tm to identify potential fragment binding. The prerequisites regarding protein quality and concentration for performing nano-DSF experiments are briefly outlined followed by a step-by-step protocol that uses a nano-liter robotic dispenser commonly used in structural biology laboratories for preparing the required samples in 96-well plates. The protocol describes how the reagent mixtures are transferred to the capillaries needed for nano-DSF measurements. In addition, this paper provides protocols to measure thermal denaturation (monitoring intrinsic tryptophan fluorescence) and aggregation (monitoring light back-scattering) and the subsequent steps for data transfer and analysis. Finally, screening experiments with three different protein targets are discussed to illustrate the use of this procedure in the context of lead discovery campaigns. The overall principle of the method described can be easily transferred to other fragment libraries or adapted to other instruments.

Monitoring changes in the melting temperature of a target protein (i.e., thermal shift assay, TSA) is an efficient method for screening fragment libraries of a few hundred compounds. We present a TSA protocol implementing robotics-assisted nano-Differential Scanning Fluorimetry (nano-DSF) for monitoring intrinsic tryptophan fluorescence and light back-scattering for fragment screening.

Thermal shift assays (TSAs) examine how the melting temperature (Tm) of a target protein changes in response to changes in its environment (e.g., buffer ...

Preparation of High-Temperature Sample Grids for Cryo-EM

The sample grids for cryo-electron microscopy (cryo-EM) experiments are usually prepared at a temperature optimal for the storage of biological samples, mostly at 4 &#176;C and occasionally at room temperature. Recently, we discovered that the protein structure solved at low temperature may not be functionally relevant, particularly for proteins from thermophilic archaea. A procedure was developed to prepare protein samples at higher temperatures (up to 70 &#176;C) for cryo-EM analysis. We showed that the structures from samples prepared at higher temperatures are functionally relevant and temperature dependent. Here we describe a detailed protocol for preparing sample grids at high temperature, using 55 &#176;C as an example. The experiment made use of a vitrification apparatus modified using an additional centrifuge tube, and samples were incubated at 55 &#176;C. The detailed procedures were fine-tuned to minimize vapor condensation and obtain a thin layer of ice on the grid. Examples of successful and unsuccessful experiments are provided.

This paper provides a detailed protocol for preparing sample grids at temperatures as high as 70 &#176;C, prior to plunge freezing for cryo-EM experiments.

The sample grids for cryo-electron microscopy (cryo-EM) experiments are usually prepared at a temperature optimal for the storage of biological samples, ...

Preparation of siRNA Mixture for Mitochondrial DNA Synthesis

Preparation of Electron Microscopy Grids for In Situ Cellular Cryotomography

Sample Preparation for In Situ Cryotomography of Mammalian Cells

In situ cellular cryotomography is a powerful technique for studying complex objects in their native frozen-hydrated cellular context, making it highly relevant to cellular biology and virology. The potential of combining cryotomography with other microscopy modalities makes it a perfect technique for integrative and correlative imaging. However, sample preparation for in situ cellular tomography is not straightforward, as cells do not readily attach and stretch over the electron microscopy grid. Additionally, the grids themselves are fragile and can break if handled too forcefully, resulting in the loss of imageable areas. The geometry of tissue culture dishes can also pose a challenge when manipulating the grids with tweezers. Here, we describe the tips and tricks to overcome these (and other) challenges and prepare good-quality samples for in situ cellular cryotomography and correlative imaging of adherent mammalian cells. With continued advances in cryomicroscopy technology, this technique holds enormous promise for advancing our understanding of complex biological systems.

Author Spotlight: Optimizing Grid Preparation for Enhanced Cryoelectron Tomography 

This method provides an accessible and flexible protocol for the preparation of electron microscopy (EM) grids for in situ cellular cryotomography and correlative light and electron microscopy (CLEM).

In situ cellular cryotomography is a powerful technique for studying complex objects in their native frozen-hydrated cellular context, making it highly ...

Quantum Dot Nanobeads for Enhanced Sensitivity in Biomarker Detection

Fluorescent Lateral Flow Immunoassay Based on Quantum Dots Nanobeads

Quantum dots, also known as semiconductor nanocrystals, are novel fluorescent labels for biological imaging and sensing. However, quantum dot-antibody conjugates with small dimensions (~10 nm), prepared through laborious purification procedures, exhibit limited sensitivity in detecting certain trace disease markers using lateral flow immunoassay strips. Herein, we present a method for the preparation of quantum dot nanobeads (QDNB) using a one-step emulsion evaporation method. Using the as-prepared QDNB, a fluorescent lateral flow immunoassay was fabricated to detect disease biomarkers using C-reactive protein (CRP) as an example. Unlike single quantum dot nanoparticles, quantum dot nanobead-antibody conjugates are more sensitive as immunoassay labels due to signal amplification by encapsulating hundreds of quantum dots in one polymer composite nanobead. Moreover, the larger size of QDNBs facilitates easier centrifugation separation when conjugating QDNBs with antibodies. The fluorescent lateral flow immunoassay based on QDNBs was fabricated, and the CRP concentration in the sample was measured in 15 min. The test results can be qualitatively assessed under UV light illumination and quantitatively measured using a fluorescent reader within 15 min.

Author Spotlight: High-Quality Quantum Dot Nanobeads for Sensitive Fluorescent Lateral Flow Immunoassays

Here, we describe a protocol for the preparation of quantum dot nanobeads (QDNB) and the detection of disease biomarkers using QDNB-based lateral flow immunoassay strips. The test results can be qualitatively assessed under UV light illumination and quantitatively measured using a fluorescent strip reader within 15 min.

Quantum dots, also known as semiconductor nanocrystals, are novel fluorescent labels for biological imaging and sensing. However, quantum dot-antibody ...