Effect of Rosemary Extract on Changes in Quality and Protein Characteristics of Penaeus Vannamei During Cold Storage

 Objective:To investigate the effect of rosemary extract (RE) on the quality change of Litopenaeus vannamei during cold storage. Methods]: The samples were macerated with 2.0% (RE1), 4.0% (RE2) and 6.0% (RE3) of rosemary extract, and the control group (CON) was macerated with sterile water. The total viable count (TVC), physical and chemical properties (pH, total volatile basic nitrogen (TVB-N), thiobarbituric acid (TBA), hardness, elasticity), protein properties (total volatile basic nitrogen (TVB-N), hardness, elasticity) were measured every 2 days. elasticity], protein properties (total sulfhydryl content and endogenous fluorescence intensity), color difference, black discoloration index and sensory analysis.

 

Results and Conclusions] Compared with the control group, rosemary extract could significantly inhibit the growth of microorganisms, slow down the increase of pH, TVB-N and TBA values, maintain good hardness and elasticity, reduce the oxidation rate of proteins, maintain the tertiary structure of proteins, and maintain the stability of the color of the surface of the shrimp during the period of cold storage. Among them, the best preservation effect was achieved by the impregnation with 4.0% rosemary extract. Compared with the control group, the impregnation of Vannamei shrimp with this concentration of rosemary extract could prolong the shelf life of the shrimp for 2-3 d. The results showed that the freshness of the shrimp could be preserved by the impregnation with 4.0% rosemary extract.

 

Litopenaeus vannamei, also known as Pacific white shrimp, South American white shrimp, is a globally important economic shrimp species for mariculture [1], which has a delicious flavor and is rich in polyunsaturated fatty acids and amino acids and other nutrients [2]. According to the China Fisheries Statistical Yearbook 2020 [3], China's production of Vannamei shrimp mariculture was 1,117,500 t, accounting for more than 77% of shrimp mariculture production. However, shrimp and other aquatic products are susceptible to spoilage during circulation due to the influence of microorganisms and endogenous enzymes, and it is therefore important to adopt appropriate treatment means to extend their shelf life. Currently, physical, chemical and biological preservation methods are commonly used for shrimp[4] . In recent years, as consumers pay more attention to food safety, biopreservation has attracted the attention of researchers because it is green and safe.

 

Bio-preservatives, also known as natural preservatives, are a wide range of safe, healthy, non-toxic food additives extracted from plants, animals and microorganisms with preservation effects. Among them, plant extracts have antioxidant and antimicrobial activities, which have been increasingly emphasized as potential natural additives. Rosemary (Rosmarinus officinalis) is a herb in the family Labiatae, and contains a variety of antioxidant components, such as silymarinic acid, geranylgeranyl alcohol, rosemary quinone, rosemary bisphenol, cinnamyl alcohol, and cinnamic acid[5] .

 

The antioxidant and antimicrobial activities of rosemary extract are mainly attributed to creatine and inositol, which contain phenolic diterpene compounds that can stabilize unsaturated fatty acids and delay their degradation, and thus have been used in aquatic products and meat products[6] . Wang Q et al.[7] found that rosemary extract had a good inhibitory effect on microbial growth in preserved Sichuan pepper meatballs, and Zhang Nannan et al.[8] used 2 g/L rosemary extract compounded with 1 g/L ε-poly(lysine) acid to treat croaker (Pseudosciaena crocea), and the iced shelf life of the croaker was extended from 6-9 d to 13-15 d. The results indicated that the microbial growth of Pseudosciaena crocea in the meatballs was inhibited by the combination of rosemary and poly(lysine) acid, which was a good inhibition of microbial growth.

 

Currently, studies on the use of biocontrol agents for shrimp preservation are mainly focused on grape seed extract [9] and pomegranate peel extract [10], but there are few reports on the use of rosemary extract for shrimp preservation in Vannamei shrimp. In this study, we investigated the effects of different concentrations of rosemary extract on the quality changes of shrimp during cold storage, and evaluated the quality changes of shrimp during cold storage through the total number of colonies, physicochemical (pH, total volatile nitrogen, TVB-N, thiobarbituric acid, TBA and texture analysis, TPA), and protein properties (total sulfhydryl group, protein fluorescence), and the color difference, black discoloration index, and organoleptic analyses, so as to provide the best results. The quality changes during cold storage were evaluated comprehensively, in order to provide theoretical references for the development and utilization of biopreservatives in shrimp aquatic products.

 

1 Materials and Methods

1.1 Main pharmaceutical reagents

TCA, ethanol, sodium chloride, magnesium oxide, purchased from Sinopharm Chemical Reagent Co., Ltd; plate counting agar, purchased from Qingdao Hi-Tech Industrial Park HaiBo Biotechnology Co. Ltd. and so on, all of which were domestic analytical pure.

 

1.2 Main instrumentation

Kjeltec8400 Kjeldahl Nitrogen Analyzer, FOSS, Sweden; H-2050R High Speed Freezing Centrifuge, Hunan Xiangyi Laboratory Instrument Development Co.

 

1.3 Raw material handling

L. vannamei was purchased from Shangyou Fresh Life Supermarket in Pudong New Area, Shanghai, China, and live shrimp with a body length of (14 ± 1) cm, body mass of (16 ± 1) g, undamaged body surface, and uniform size were selected. The shrimp were kept alive in an oxygen-filled transportation box and transported to the laboratory within 30 min.  The shrimps were inactivated by crushed ice, washed and drained with water and then randomly divided into four groups, which were treated with sterile water (CON), 2.0%, 4.0% and 6.0% rosemary extract solution for 10 min, with the ratio of shrimp to water being 1:2, and then drained naturally for 15 min, and then placed in a plastic bag in a refrigerator at (4 ± 1) ℃, and the indexes were determined every 2 d. The shrimps were then stored in the refrigerator at (4 ± 1) ℃.

 

1.4 Experimental Methods

1.4.1 Total Viable Counts (TVC)   

 

The method of GB 4789.2-2016 [11] was used to determine the total number of colonies. Weigh 5 g of shrimp meat into a sterile homogenization bag containing 45 mL of 0.85% sterile saline, homogenized and then diluted in a gradient. The results were counted after 48 h of incubation at 30 ℃ in a constant temperature incubator using the pouring and inverting plate method, and the results were expressed as the logarithm of the total number of colonies.

 

1.4.2 Physical and chemical indicators

1.4.2.1 pH  

The pH value was determined by the method of GB 5009.237-2016 [12]. Shrimp meat was mixed with distilled water at a mass ratio of 1:9, and then left to stand for 30 min, and the pH value was measured by a pH meter.

 

1.4.2.2 Total volatile basic nitrogen (TVB-N)   

The TVB-N values of each group of samples were determined during storage according to the automatic Kjeldahl method of GB 5009.228-2016[13] .

 

1.4.2.3 Thiobarbituric acid (TBA)

Referring to the method of Ge et al [14], 5 g of shrimp meat was weighed and mixed with 25 mL of 20% TCA solution by volume for 1 min, centrifuged at 8000 r/min for 10 min, and the supernatant was concentrated to 50 mL and then shaken well. Take 5 mL of the above solution, mix with 5 mL of 0.02 mol/L thiobarbituric acid solution, then react with boiling water for 20 min, cool down to room temperature with running water, and absorb the color-developing solution to determine the optical density at 532 nm, D. Calculate the TBA value according to the TBA value = 7.8×D, and the unit is mg/kg.

 

1.4.2.4 Texture Profile Analysis (TPA)

With a slight modification of the method of Liu et al. [15], the second abdominal muscle of shrimp was taken and a 6 mm diameter flat-bottomed cylindrical probe was used as follows: pre-test rate of 4 mm/s, test rate of 1 mm/s, post-test rate of 5 mm/s, compression rate of 50%, compression interval of 5.0 s, and trigger value of 15 g. Hardness and elasticity values of the samples were recorded for each sample group at different storage times. The hardness and elasticity values were recorded for each group of samples at different storage times.

 

1.4.3 Protein characterization

1.4.3.1 Total sulfhydryl content   

The optical density at 412 nm was determined spectrophotometrically according to the instructions of the total sulfhydryl content determination kit. The molecular extinction coefficient was taken as 13 600 L/(mol-cm).

 

1.4.3.2 Endogenous fluorescence intensity    

Myofibrillar protein was extracted by the method of Li et al. 2 g of shrimp meat was homogenized with 20 mL of buffer (20 mmol/L Tris-maleate, pH 7.0, 0.05 mol/L KCl), and then centrifuged at 10 000 r/min for 15 min at 4 ℃, leaving a layer of starch that was mixed with 20 mL of buffer (20 mmol/L Tris-maleate, pH 7.0, 0.05 mol/L KCl), and a layer of starch that was mixed with 20 mL of buffer (20 mmol/L Tris-maleate, pH 7.0, 0.05 mol/L KCl).

The extract was homogenized with 0.6 mol/L KCl and left at 4 ℃ for 1 h. The mixture was centrifuged at 10 000 r/min for 15 min at 4 ℃, and the supernatant was myofibrillar protein solution, which was stored at 0-4 ℃ for 1 h. The extracted myofibrillar protein solution was subjected to endogenous fluorescence high-speed scanning detection. The extracted myofibrillar protein solution was subjected to endogenous fluorescence high-speed scanning detection with the following parameters: excitation wavelength of 295 nm; emission wavelength of 300~400 nm; scanning speed of 1 200 nm/min; and slit width of 5 nm.

 

1.4.4 Color difference  

After calibrating the colorimeter with a white calibration plate according to the method of Wang Chunling et al [17], the luminance L*, redness a* and yellowness b* were measured on the surface of the second abdominal segment.

1.4.5 Black change index  

The black discoloration of the different groups of samples was scored by six professionally trained persons according to the method of Kim et al [18] using the scoring criteria in Table 1. The smaller the score, the higher the freshness.

 

1.4.6 Sensory evaluation  

The overall acceptability of Shrimp Vannamei was assessed using a 9-point scale based on the method of Azizi-lalabadi et al [19]. A sensory panel of six trained personnel was formed and the overall score was based on the sensory evaluation of the samples in terms of color, odor and texture. The samples were categorized into four grades: bad or unacceptable (1-3 points), good (4-6 points), very good (7-8 points) and excellent (9 points).

 

1.5 Data processing

All experiments were repeated three times, and the data were statistically analyzed by SPSS17.0 software, plotted by Origin85 software, and analyzed by one-way ANOVA with a significance level of α = 0.05. The results of the analysis of variance (ANOVA) were obtained by using a one-way ANOVA.

 

2 Results and analysis

2.1 Total colony count

The changes in the total number of colonies of Penaeus vannamei during cold storage in the presence of different rosemary extracts are shown in Fig. 1.

From Fig. 1, it can be seen that the total colony counts of the CON and RE treatment groups showed an increasing trend with the increase of storage time, the initial logarithmic value of the total colony count of the CON group was 2.67±0.09, and the logarithmic value of the total colony count of the CON group reached 7.56±0.08 after the whole storage period, and the total colony counts of the RE group were significantly reduced compared with the total colony counts of the CON group in the 8 d of storage period (P<0.05), with a range from 0.41 to 1.75. The logarithmic values of total colony count of RE1 and RE2 samples did not exceed 7 throughout the storage period. Compared with the CON group, the total number of colonies in both RE treatment groups decreased significantly (P < 0.05), ranging from 0.41 to 1.75, whereas the logarithmic value of the total number of colonies in the RE1 group and RE2 samples never exceeded 7 during the whole storage period.

 

The lowest values of total colony counts were found in RE3, which showed that the dipping treatments with rosemary extract at 4.0% and 6.0% by mass had a significant inhibitory effect on the total colony counts during storage of Penaeus vannamei. This result is in line with the results of Nawaz et al [20] on the preservation of pangolin (Cirrhinus mrigala) by rosemary extract. Hussein et al [21] reported that rosemary extract delayed the growth of Cryptophilus spp. and elevation of biogenic amines during cold storage of chicken cutlets.

 

2.2 pH and TVB-N Values

Total volatile salt base nitrogen (TVB-N) in shrimp meat refers to the breakdown of proteins into alkaline compounds such as ammonia and amines by microorganisms and enzymes [22]. The effect of different concentrations of rosemary extracts on the pH and TVB-N values of shrimp Penaeus vannamei during cold storage is shown in Figure 2.

As shown in Fig. 2A, the pH values of the groups showed an increasing trend with storage time, which may be related to the accumulation of volatile alkaline compounds[23] . Compared with the CON group, the pH values of the RE2 and RE3 groups increased more slowly at the beginning of storage. Gao et al [24] found that rosemary extract could slow down the increase in pH value of chicken breast meat during storage, which is consistent with the results of this study. This may be related to the fact that rosemary extract inhibited the growth of spoilage bacteria and reduced the production of amines; on the other hand, the phenolic acid compounds such as rhamnoside, rosemary acid, caffeic acid and ursolic acid were found in the extract, which were effective in inhibiting the increase of pH value [25].

 

From Fig. 2B, it can be seen that the TVB-N values of each treatment group tended to increase with the prolongation of refrigeration time. For 2 d of storage, the TVB-N values of sterile water and RE treatment groups were similar, (11.15 ± 0.73), (10.01 ± 0.55), (8.98 ± 0.23), (8.76 ± 0.49) mg/100 g. From 4 d onwards, the differences in TVB-N values of samples from each treatment group were as follows

The rate of increase of TVB-N value in shrimp samples of each group was significant (P＜0.05), and the rate of increase of TVB-N value in shrimp samples of each group was as follows: CON group > RE1 group > RE2 group > RE3 group. The rate of increase of TVB-N value in the control group was significantly faster than that in the RE-treated group. At 6 d of storage, the TVB-N value of the CON group was (32.66 ± 0.36) mg/100 g, which exceeded the spoilage limit (30 mg/100 g), while the TVB-N values of the RE1, RE2 and RE3 groups were (27.78 ± 0.55), (24.05 ± 0.51) and (21.90 ± 0.66) mg/100 g, respectively. The results showed that rosemary extracts impregnated with 4% and 6% by mass were more effective in preserving the freshness of the shrimp, because they could inhibit microbial growth, reduce the rate of proteolysis, and decrease the accumulation of nitrogenous compounds in shrimp meat.

 

2.3 TBA value and total sulfhydryl content

Oxidation of unsaturated fatty acids in shrimp is one of the main causes of off-flavors, and the TBA value is an important indicator of the degree of oxidation of fats, with higher levels of oxidation resulting in higher levels of oxidized products such as aldehydes and ketones.

 

Fig. 3A shows that the TBA values of shrimp Vannamei treated with different concentrations of rosemary extract showed a general increasing trend. In the pre-storage period, the differences in TBA values between different treatments were not significant (P > 0.05), but with the extension of storage time, the differences were significant (P< 0.05). The initial TBA value of the CON group was 0.029 mg/kg, and it was 0.69 mg/kg at 8 d of storage, whereas the TBA values of the RE1, RE2, and RE3 groups were 15.9%, 24.6%, and 20.3% lower than those of the CON group. 15.9%, 24.6% and 20.3% lower than the TBA value of the CON group in the RE1, RE2 and RE3 groups, respectively. This may be attributed to the antioxidant property of rosemary extract, which reduces the rate of fat oxidation and the production of unsaturated fatty acid oxidation products.  Jafari et al [26] found that the silymarinic acid in rosemary extract provided phenolic hydrogens, which led to the formation of stabilized end-products and altered the free radical chain. Because of the strong chelating capacity of metal ions, the rosemary extracts, including rhamnolic acid and rosemarinol, can effectively stabilize free radicals and thus inhibit the fat oxidation of shrimp meat [27].

 

The total sulfhydryl content is one of the indicators of the degree of protein oxidation. As shown in Figure 3B, the total sulfhydryl content of the samples decreased during 8 d of refrigeration, which was attributed to the oxidation of the reactive sulfhydryl groups inside the protein molecules by exposure. The decrease in total sulfhydryl content slowed down with increasing concentrations of rosemary extract (P < 0.05), suggesting that the rosemary extract retarded the oxidation of shrimp myofibrillar proteins. The total sulfhydryl mass molar concentration was (2.15 ± 0.04) μmol/g at 0 d, and decreased to (1.12 ± 0.07), (1.24 ± 0.05), (1.32 ± 0.03), and (1.42 ± 0.04) μmol/g for samples of RE1, RE2, and RE3 groups, respectively, at 8 d of refrigeration. Nie et al. [28] investigated the changes of total sulfhydryl groups in the muscle of sea bass (Lateolabrax japonicas) during refrigeration, which is consistent with the results of the present study, and it may be attributed to the oxidation of sulfhydryl groups to disulfide bonds, which resulted in the decrease of total sulfhydryl groups. The difference between the CON group and the treated group was significant (P<0.05). It can be concluded that the rosemary extract can inhibit protein oxidation during storage.

 

2.4 Texture analysis

As shown in Fig. 4, the hardness of the different treatments of shrimp Vannamei showed a decreasing trend during refrigeration, and at 8 d, the hardness of the samples in groups CON, RE1, RE2 and RE3 decreased to 28.01%, 48.38%, 52.24% and 50.66% of the initial hardness value of the fresh samples (897.12±8.72) (Fig. 4A), respectively. It is possible that protein degradation and loss of juice in shrimp meat lead to structural changes in myofibrillar proteins [29]. Elasticity indicates the rate of deformation of shrimp meat due to external force and recovery after withdrawal of the force. The elasticity values (Fig. 4B) of the samples in each group decreased with the increase in freezing time due to the reduction in the structural toughness of the muscle tissue as a result of microbial and endogenous proteolytic enzymes[30] . Compared with the CON group, the samples from the RE2 and RE3 groups showed a smaller decrease, which is consistent with the trend of the elasticity changes of rosemary extract on ice-frozen yellow croaker studied by Lan et al [31]. This is consistent with the trend of rosemary extract on the elasticity of ice-frozen croaker studied by Lan et al [31], which indicated that rosemary extract could slow down the rate of protein degradation and stabilize the muscle structure of shrimp.

 

2.5 Endogenous fluorescence spectroscopy

Tryptophan residues have fluorescent properties, and the fluorescence emitted from them by absorbing UV light is very sensitive to the polarity of the surrounding microenvironment [32]. When the protein is folded, the tryptophan residues are located in the hydrophobic environment inside the protein and emit high fluorescence intensity after excitation; when the protein is fully or partially unfolded, when the tryptophan residues are exposed on the surface of the protein, the fluorescence intensity decreases after excitation. Therefore, the endogenous fluorescence intensity can be monitored to study the conformational changes of proteins [33].

As shown in Fig. 5, the highest fluorescence intensity is found in the fresh sample, with the highest emission at 334 nm. With the increase of storage time, the natural arrangement of amino acids was changed, and the endogenous tryptophan residues were gradually exposed to the polar environment, and the fluorescence intensity decreased. Compared with the CON group, the endogenous fluorescence intensity of the samples from the RE1, RE2 and RE3 groups decreased less, indicating that the rosemary extract could maintain the tertiary structure of the proteins during storage.  Shi et al. [34] found that the glazed extract of rosemary had a protective effect on the protein structure of mud shrimp (Solenocera melantho).

 

2.6 Color difference

Color is a visual basis for consumers to judge the freshness of aquatic products [35]. Figure 6 shows the effect of different treatments on the changes of L*, a* and b* values of the muscle of frozen shrimp Penaeus vannamei.

As shown in Figure 6A, the L* values of the samples decreased with the storage time, indicating that the muscle brightness decreased. Compared with the samples in the CON group, the treatment with different concentrations of rosemary extract could delay the decrease of L* value and stabilize the color better, which is consistent with the study of Huang et al. [36], who investigated that rosemary extract improved the brightness of minced pork. The a* values of the samples with different treatments (Fig. 6B) showed an increasing trend, which may be attributed to the darkening of the shrimp body due to the action of polyphenol oxidase, and the darkening of the shrimp body color. The a* value of the CON group reached a positive value at the end of storage, which was significantly higher than that of the RE group. The a* values of the CON group reached a positive value at the end of storage, which was significantly higher than that of the RE group. The a* values of the CON group were significantly higher than those of the RE group. The b* values of the samples (Fig. 6C) increased with the increase of storage time, and the b* values of the RE3 group increased significantly due to the effect of the color of the extracts, while the lower concentration of the samples of the RE2 group did not have any negative effect on the color of shrimp during the storage period, and it could prolong the shelf life of shrimp while maintaining the stability of the color of its body surface.

 

2.7 Black Change Value and Sensory Score

As a result of polyphenol oxidase catalysis, colorless phenolics are oxidized to quinones, which undergo a series of biochemical reactions to produce black substances [37].

As shown in Fig. 7A, the blackening scores of all groups of samples increased significantly (P < 0.05) during the refrigeration period. The blackening of the shrimp body was delayed by the rosemary extract treatment compared to the CON group. The blackening scores of samples from RE2 and RE3 groups were significantly lower than those of RE1 group. As can be seen from the figure, black spots appeared on the cephalothorax of shrimp samples from the CON and RE1 groups at 2 and 4 d, respectively, while black spots appeared at 6 d in the RE2 and RE3 groups.

As shown in Fig. 7B, the organoleptic scores of Vannamei shrimp decreased during the storage period, which indicated that the quality of Vannamei shrimp was decreasing, and the organoleptic scores of the samples from the RE2 group were higher than those of the other treatment groups during the storage period, which were significantly different from those of the control group (P<0.05), and the samples also showed better quality characteristics in terms of appearance and flavor. This indicated that the best sensory scores were obtained at 4% extract mass fraction, and the sensory scores decreased with increasing concentration.  Hao et al. [38] found that the addition of a treatment with a mixture of dieperidin extracts was effective in maintaining the good organoleptic qualities of abalone (Haliotis discus Hannai Ino).

 

3 Conclusion

Rosemary extract impregnation significantly inhibited the growth of microorganisms, slowed down the increase of pH, TVB-N and TBA values, maintained the hardness and elasticity of shrimp meat, lowered the oxidation rate of proteins, maintained the stability of the tertiary structure of proteins, and stabilized the color of the body surface of the samples during the storage period. The best preservation effect was achieved by impregnation with 4.0% rosemary extract. Compared with the control group, the dipping of shrimp Vannamei at this concentration extended the shelf-life of the samples for 2~3 d. The results showed that the dipping of shrimp Vannamei at this concentration extended the shelf-life of the samples.

 

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