Preparation of High-Quality Fermented Fish Product

Summary

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

Abstract

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 °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 °C for 6-10 h; (5) sealing fish cubes in vacuum packages with marinating and fermenting solutions; (6) sterilizing at 115-121 °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.

Introduction

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 researchers^1,2,3,4. 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 products^8,9,10 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 oxidation^11,12. 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 studies^15,16,17, 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.

Protocol

1. Sample preparation

1. Thaw frozen sturgeon meat under flowing water below 20 °C. Then, clean, skin off and cut the fish into cubes (2 cm × 2 cm × 1.5 cm).
   NOTE: Raw material could be fresh or frozen sturgeon meat.
2. Mix fish cubes with 6-12% (w/v) salt solution at a rate of 1:1 (fish cube mass to solution volume), at 10 °C for 1-3 h.
3. Put the marinated fish cubes onto a stainless-steel gridding plate and dry fish cubes at 40-60 °C for about 6-10 h by hot air, or dry the fish cubes in vacuum, until the water content decreases to 50-60%.
4. 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 °C for 6-10 h.
   NOTE: The fermentation must be performed under anaerobic environment.
   1. 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.
   2. 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.
5. 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.
6. 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 °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 °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.

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.

1. Store products at 20 °C, 30 °C, 40 °C.
2. Predict the shelf life by the Arrhenius model using rancidity rates. Measure rancidity rates by testing acid values (AV) under different temperatures.
3. Measure AV on the 0^th, 14^th, 28^th, 42^nd, 56^th, 70^th days.

3. Chemical analysis

1. Determination of salt content
   NOTE: We measure salt content according to the method proposed by Zeng et al.¹⁹.
   1. Accurately weigh 1 g of the sample and add 10 mL of 0.1 M AgNO₃ + 10 mL of HNO₃.
   2. Heat the mixture gently on an induction-cooker for 10 min.
   3. Cool the mixture with running water, and add distilled water (50 mL) and ferric alum indicator (5 mL).
   4. Titrate the mixture with standard 0.1 M KSCN until the solution turns permanent brownish-red.
      NOTE: Calculate and express the results as % NaCl.
2. Determination of moisture content
   1. Determine moisture content according to the method of Zeng et al.¹⁹.
3. Determination of pH value
   NOTE: We measure pH value according to the method of Zeng et al.⁶.
   1. Homogenize 10 g of the sample with 90 mL of deionized water.
   2. Measure the pH value with a digital pH meter.

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.

1. Extraction of volatile flavors
   1. Weigh 2 g of the sample accurately, and then put it into the sample vial.
   2. 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.
   3. Put the sample vial on the magnetic stirrer and stir for 10 min to mix the sample homogenously.
   4. Insert the fiber into the headspace of the sample vial.
   5. Adsorb at 60 °C for 30 min, and then desorb at 250 °C for 3 min in the injection port.
2. Analysis of flavor by GC-MS
   1. Refer to the Table of Materials for details of the instrument used.
   2. Use Helium (purity > 99.995%) as the carrier gas, and set the flow rate to 0.9 mL/min.
   3. Set the column temperature to 40 °C for 3 min initially, and then increase to 90 °C at a rate of 5 °C/min. Subsequently, increase to 230 °C at a rate of 10 °C/min.
   4. 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 μA. Use interface and source temperatures of 250 and 200 °C, respectively.
   5. 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).
   6. 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 μg/kg.

5. Texture profile analysis

NOTE: Analyze texture profile by following a previous study²⁰.

1. Perform texture profile analysis (TPA) with a texture analyzer equipped with a cylindrical probe (P/36R).
2. Apply two consecutive cycles. Deformation degree of TPA is 50% and the trigger force is 5 x g.
3. 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.

6. Color measurement

NOTE: Measure color according to the method of Czerner et al. with some modifications²¹.

1. Measure samples by using colorimeter. Record the lightness value (L*), greenness/redness value (a*) and blueness/yellowness value (b*) of the samples.
2. Measure five replicates for each sample.

7. Sensory evaluation

1. 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.
2. Score the samples from 0 to 10 for taste, flavor, color and texture. Score 0 represents "dislike extremely" and score 10 represents for "like extremely".
   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.

Quality parameter	Description	Score
Taste	Acceptable sweet and salty; harmonious taste; wine taste; not astringent	8-10
	Too heavy or light (sweet or salty); harmonious taste; wine taste; not astringent	6-8
	Too heavy or light (sweet or salty); light wine- and after- taste; astringent	3-6
	Too light wine- and after- taste; obviously astringent	0-3
Flavor	Mellow wine flavor; rich fermentation aroma; no weird smell	8-10
	Mellow wine flavor; light fermentation aroma; no weird smell	6-8
	Light wine flavor; light fermentation aroma; light weird smell	3-6
	No wine aroma; light fermentation aroma; obviously weird smell	0-3
Appearance	Russet color; glossy appearance; bright surface	8-10
	Russet color; bright surface	6-8
	Yellow color; nonuniform	3-6
	Pale color; rough surface	0-3
Texture	Acceptable chewiness	8-10
	Hard	6-8
	Light springiness	3-6
	Soft texture; coarse mouth-feel	0-3

Table 1: Scoring criteria for sensory quality of fermented wine-aroma sturgeon meat product.

Results

The suitable salt concentration, marinating time and temperature make the product’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 °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...

Discussion

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 ...

Materials

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