Abstract: In this study, the endogenous fluorescence intensity, sulfhydryl content, rheological properties, gel strength and water retention of myofibrillar fibrillar proteins from porcine longissimus dorsi muscle were investigated to investigate the effects of rosemary extract (0.01 g/g pro) on myofibrillar fibrillar proteins' structure and gel properties under different NaCl concentration conditions. The results showed that the addition of rosemary extract to myofibrillar fibrillar proteins at NaCl concentrations lower than 0.45 mol/L was not conducive to the formation of a good gel structure, and the water retention of myofibrillar fibrillar proteins was poor; however, at a NaCl concentration of 0.45 mol/L, the addition of rosemary extract together with NaCl could significantly improve the energy storage modulus, gel strength and water retention of myofibrillar fibrillar proteins. At this NaCl concentration, the synergistic effect of rosemary extract and NaCl significantly decreased the endogenous fluorescence intensity of myofibrillar fibrillar proteins, and the wavelength was red-shifted by 2 nm. The favorable change in the tertiary structure of myofibrillar fibrillar proteins was an important reason for the lack of a significant decrease in water retention of myofibrillar fibrillar proteins under the reduced-salt condition. The above results may provide a new reference for the development of NaCl substitutes.
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Myofibrillar protein (MP) is a salt-soluble protein, which is the main component of muscle proteins, and its main functional property is the ability to thermally induce the formation of gels, which gives meat products, especially minced meat, a good texture and taste. Solubility is closely related to the emulsifying gel properties of MP. Within a certain ionic strength range, the solubility increases with the increase of ionic strength, therefore, NaCl is usually added to increase the ionic strength in meat processing, and the solubility of MP is increased by chopping and mixing. It has been shown that the gel strength of pork MP increases with the increase of ionic strength within a certain ionic strength range[1] .At a NaCl concentration of 0.6 mol/L, the solubility of MP is high, and it is able to form a regular and orderly three-dimensional gel network structure, whereas at a low concentration of NaCl, most of the MP is in the unresolved state, and it is not able to form a stable gel structure[2] .
Spices are the main auxiliary materials in meat processing, and their use can give certain flavor to the products on the one hand, and play the role of antioxidant and antibacterial on the other hand. Currently, there are many research reports on the antimicrobial and antioxidant effects of spice extracts in meat products. The application of rosemary and bee balm extracts in cooked ground pork products significantly reduced the thiobarbituric acid value and hexanal content, suggesting that they can inhibit fat oxidation [3-4]. As two major auxiliary materials in meat processing, NaCl and spices may affect the structural and functional properties of proteins to some extent; however, how they synergistically affect the structural and functional properties of MPs, and thus the quality of minced meat products, is not fully understood. In the present study, mixtures of pork MP with different concentrations of NaCl and rosemary extract were prepared, and the sulfhydryl content, endogenous fluorescence intensity, rheological properties, gel strength, and water retention were measured to investigate the effects of NaCl concentration and rosemary extract on the structure and gel properties of MP.
1 Materials and Methods
1.1 Materials and reagents
The longest muscle of Saintyear pig was purchased from Jinzhou RT-Mart Supermarket; rosemary was purchased from local pharmacy.
Sodium chloride, sodium dihydrogen phosphate, disodium hydrogen phosphate, ethanol, sodium phosphate, magnesium chloride, ethylenediaminetetraacetic acid (EDTA), etc. are domestic analytical purity; Tris American Sigma Company.
1.2 Instruments and equipment
FW-2000 High-speed Universal Pulverizer Beijing Zhongxing Weiye Instrument Co., Ltd; RE-52AA Rotary Evaporator Shanghai Yarong Biochemical Instrument Factory; THZ-100B Constant Temperature Cultivation Shaker Shanghai Yiheng Scientific Instrument Co. Ltd; T25 Digital Homogenizer, RCT Basic Magnetic Stirrer, IKA Group, Germany; PL602-L Electronic Balance, Shanghai Sizhan Measuring Instrument Company Limited; 970CRT Fluorescence Spectrophotometer, Shanghai Precision Scientific Instrument Company Limited; UV-2550 Ultraviolet-Visible Spectrophotometer, Shimadzu Instruments (Suzhou) Co. Hunan Xiangyi Laboratory Instrument Development Co., Ltd; TA-XT Plus Texture Analyzer, Stable Micro Systems, UK; DHR-1 Rheometer, TA Instruments, USA.
1.3 Methodology
1.3.1 Preparation of rosemary extracts
Refer to the method of Zhang Xue et al[5] with appropriate modifications. Rosemary was washed and drained, dried in a drying oven at 45 ℃, crushed with a high-speed universal pulverizer, and passed through a 40-mesh sieve. Take 50 g of powder in a 1 000 mL stoppered conical flask, add 400 mL of 95% ethanol, stopper the flask tightly, and place it in a constant temperature incubation shaker, extract it at 55 ℃ and 110 r/min for 12 h. Filter it, and add 200 mL of 95% ethanol to the residue to re-extract it for 12 h, and filter it, and combine the filtrates, and then concentrate the concentrated liquid under vacuum in a rotary evaporator for 2 h (55 ℃, 0.08 MPa), and then concentrate the concentrated liquid at -50 ℃. The concentrated liquid was freeze-dried under vacuum at -50 ℃ under a vacuum of 7 Pa. The extract was stored at -20 ℃ for spare parts (rosemary extraction rate of 21.28%, water content of 5%).
1.3.2 Extraction of MP and sample preparation
The method of Liu Gang et al[6] was used with slight modification. About 500 g of pork was weighed, crushed, and 4 times the volume of extraction solution (10 mmol/L Na3PO4, 0.1 mol/L NaCl, 2 mmol/L MgCl2, and 1 mmol/L EDTA, pH 7.0) was added, homogenized for 60 s, and then centrifuged at 3,500 r/min for 15 min, and then the precipitates were extracted by repeating the above steps twice, and then the precipitates were added with 4 times the volume of 0.1 mol/L NaCl solution, and washed three times according to the above conditions. Add 4 times the volume of 0.1 mol/L NaCl solution, wash the precipitate 3 times according to the above centrifugation conditions, filter with 4 layers of gauze before the last centrifugation, and then adjust the pH to 6.0 with 0.1 mol/L HCl solution, and then refrigerate the obtained MP at 4 ℃ for spare use. The content of MP was determined by the bis-urea method using bovine serum albumin as the standard.
The MP solutions were prepared at concentrations of 5 mg/mL and 40 mg/mL by adding 0.01 g/g pro of rosemary extract, and then adding NaCl to the mixtures to achieve final concentrations of 0.00, 0.15, 0.45, and 0.60 mol/L, respectively. The prepared MP mixture was refrigerated at 4 ℃ overnight (12h). The prepared MP mixture was refrigerated at 4 ℃ overnight (12 h), and the MP without rosemary extract was used as the control for the subsequent determination of various indexes.
1.3.3 Determination of the mass molar concentration of sulfhydryl groups
Referring to the method of di Simplicio et al[7] and slightly improved, take 1 mL of the above 5 mg/mL MP solution, add 8 mL Tris-glycine solution to dissolve it, homogenize it and centrifuge it at 10 000 r/min for 15 min, then remove the insoluble proteins and take 4.5 mL of the supernatant, add 0.5 mL of 10 mmol/L Ellman's reagent, and then let it stand for 30 min after shaking well. After 30 min, the absorbance was measured at 412 nm, and phosphate buffer solution was used as control. The mass molar concentration of sulfhydryl groups was calculated using the molar extinction coefficient method, and the resulting mass molar concentration of sulfhydryl groups was expressed as μmol/g pro, which was calculated as shown in Equation (1).
Molar concentration of sulfhydryl mass/ (µmol/g pro) = x D
(1) Where: A is the absorbance; ε is the molar extinction coefficient (13 600 L/ (mol -cm )); D is the dilution.
1.3.4 Measurement of endogenous fluorescence intensity
The method was based on that of Li Xuepeng et al [8] with appropriate modifications. 0.5 mL of 5 mg/mL MP solution was accurately measured and diluted to 0.1 mg/mL with 50 mmol/L phosphate buffer at pH 7.0, and then centrifuged at 10 000 r/min for 30 min with magnetic stirring for 2 h. The results were analyzed by fluorescence spectrophotometer. The parameters were set as follows: the excitation wavelength was 295 nm, the wavelength scanning range was 300-400 nm, the scanning rate was 12 000 nm/min, the excitation and emission slit widths were 2.5 nm, and the sensitivity was 3.
1.3.5 Preparation of MP gels
Take 15 mL of 40 mg/mL MP solution treated according to section 1.3.2, put it in a weighing bottle, seal it, and put it in a 70 ℃ thermostatic water bath, heat it continuously for 30 min, and then cool it down with tap water for 30 min, and then store the gel sample in a refrigerator at 2～4 ℃ for spare. The gel was stored in a refrigerator at 2~4 ℃. The MP gel was placed at room temperature for 20 min before measurement.
1.3.6 Determination of gel strength
The determination was carried out using a TA-XT Plus mass spectrometer. The samples to be tested were placed on the platform with the weighing vials, and the parameters were: probe type P/0.5, downward pressure distance of 50% of the gel height, trigger force of 5 g, pre-test rate of 2 mm/s, test rate of 1 mm/s, and post-test rate of 2 mm/s. The results were analyzed by the TA-XT Plus.
1.3.7 Determination of water retention properties
The method of Kocher et al.[9] was used with appropriate modifications. Weigh 5 g of gel sample, put it in a 15 mL centrifuge tube, weigh the mass as m2/g, then centrifuge it at 3000×g for 15 min, remove the water and weigh the mass again as m1/g, and the mass of the centrifuge tube is recorded as m0/g, and the water retention is calculated according to equation (2).
Water retention × 100
1.3.8 Determination of dynamic rheological properties
Take a certain amount of the prepared MP solution, measured by rheometer, selected 50 mm plate test, the MP emulsion under different treatment conditions uniformly spread on the test platform to eliminate air bubbles and film silicone oil sealing, the test parameters as follows
The energy storage modulus (G') was determined at a frequency of 0.1 Hz, a strain of 2%, a clamp spacing of 0.5 mm, a starting temperature of 20 ℃, a temperature increase rate of 1 ℃/min, and an ending temperature of 80 ℃.
1.4 Statistical analysis of data
Each experiment was repeated 3 times and the results were expressed as ± s . Data were analyzed statistically using the Linear Models program in SPSS 19.0 software, and the significance of differences was analyzed using the LSD method, with P < 0.05 considered significant. SigmaPlot 12.0 software was used for plotting.
2 Results and analysis
2.1 Effect of NaCl concentration and rosemary extract on MP structure
Proteins have a specific spatial structure in the natural state, and the tryptophan in proteins is buried in the internal hydrophobic environment, so the endogenous fluorescence intensity of tryptophan is higher and the fluorescence emission wavelength is shorter; however, in the case of partial unfolding, the fluorescence intensity of proteins decreases and the emission wavelength is shifted by a red shift [10]. As can be seen in Figure 1, the endogenous fluorescence intensity of the rosemary extract group and the control group did not change regularly with the increase of NaCl concentration, and the specific reason needs to be further investigated; when the NaCl concentration was 0.45 mol/L, the emission wavelength of tryptophan in the rosemary extract group was red-shifted from 332 nm to 334 nm, and the intensity of endogenous fluorescence was decreased compared with that in the control group.
The decrease in the endogenous fluorescence intensity of tryptophan could come from the partial de-helicalization of the rod tail of myosin [11] or from the structural changes in the head of myosin (the rod tail contributes only 27% of the total fluorescence intensity of myosin) [12-13]. (the contribution of the rod tails to the fluorescence intensity of myosin molecules is only 27% of the total amount) [12-13], and whether the MP tertiary structure changes induced by rosemary extracts originate from the head or the rod tails of myosin needs to be further confirmed. In addition, the polyphenols and terpenoids in rosemary extract [14] may also affect the endogenous fluorescence intensity of MP through a "shielding effect", which can be explained by the "hand and glove" model (some of the hydrophobic groups in the protein form a "glove", and some of the hydrophobes in the protein form a "glove", which can be used as a "shielding effect"). This effect can be explained by the "hand and glove" model (some hydrophobic groups in proteins form a "glove", while polyphenols bind to proteins through hydrogen bonding to form a "hand", and this shielding effect may lead to a decrease in fluorescence intensity[15] ). The "shielding effect" may also be reflected in the binding of phenolic substances in rosemary extract to proteins and adsorption on the surface of proteins, which may play a certain protective effect on proteins and lead to a decrease in fluorescence intensity [16].
2.2 Effect of NaCl concentration and rosemary extract on mass molar concentration of sulfhydryl groups
Sulfhydryl group is an important active group in MP, which can be easily oxidized to form disulfide bonds, causing intermolecular cross-linking and polymerization of proteins, and thus affecting the functional properties of proteins [17]. As shown in Figure 2, when the NaCl concentration was increased from 0.15 mol/L to 0.60 mol/L, the molar concentration of sulfhydryl groups in MP with or without rosemary extract did not change significantly (P＞0.05), indicating that increasing the ionic strength did not have a significant effect on the molar concentration of sulfhydryl groups in MP. At a certain NaCl concentration, the addition of rosemary extract resulted in a significant decrease (P<0.05) in the molar concentration of sulfhydryl groups compared with the control. This may be attributed to the fact that the main components of rosemary extract are salvia divinorum and salvia divinorum acid, both of which contain a sulfhydryl-binding site, resulting in a decrease in the sulfhydryl content.18-19 Prodpran et al.[20] obtained a similar result when investigating the effect of polyphenols on the total sulfhydryl content of MP in fish, i.e., phenolic compounds led to a decrease in the total sulfhydryl content, possibly due to the interaction between sulfhydryl and phenolic hydroxyl groups to form a more stable and stable sulfhydryl group. hydroxyl groups interact with each other to form a more stable conformation. In addition, high concentrations of green tea polyphenols can also form sulfhydryl-quinone adducts with MP sulfhydryl groups through covalent cross-linking, preventing the formation of stable disulfide bonds between proteins [21].
2.3 Effect of NaCl concentration and rosemary extract on gel strength
As can be seen in Figure 3, the gel strength of the MP+rosemary and MP groups did not change significantly (P＞0.05) when the NaCl concentration was increased from 0.15 mol/L to 0.60 mol/L. (P＞0.05), but when the NaCl concentration increased from 0.15 mol/L to 0.60 mol/L, the gel strength of both groups increased significantly (P＜0.05). This is due to the fact that with the increase of NaCl concentration, the MP and NaCl ions interacted to form a double electron layer of ionic groups, which decreased the electrostatic interactions between MP molecules, increased the repulsive force between MP molecules, and enhanced the hydration, thus improving the gel properties [22-23]. In addition, the increase of repulsive force between MP molecules led to the swelling of MP and the increase of water holding capacity, which is consistent with the results of water retention [24]. When the NaCl concentration was 0.00 and 0.15 mol/L, the gel strength of the MP+rosemary group was not significantly different from that of the control group (P＞0.05), which may be attributed to the fact that the full play of the MP gel properties requires the provision of sufficient solubility, and the solubility of MP was poor when the NaCl concentration was low (0.00 and 0.15 mol/L).
Under the high solubility condition (0.45, 0.60 mol/L), rosemary extract could improve the gel strength of MP, but different results were obtained under different NaCl concentrations. The gel strength of the MP+rosemary group was significantly higher than that of the MP group at a NaCl concentration of 0.45 mol/L (P<0.05), which may be attributed to the fact that polyphenolic substances contained in rosemary extract could easily form o-benzoquinone or o-benzohemiquinone, which could react with the sulfhydryl groups and amino groups in protein by nucleophilic addition, resulting in the formation of C-N or C-Hexaquinone between protein and phenol. This may be due to the fact that under this condition, the polyphenols contained in the rosemary extract can easily form o-benzoquinone or o-benzoquinone, which can react with the sulfhydryl and amino groups in proteins in nucleophilic addition reactions, resulting in the formation of C-N or C-S covalent bonds between proteins and phenols [25-28]. The increase in ionic strength may have hindered the formation of some protein-polyphenol covalent bonds. It can be seen that the significant effect of rosemary extract on the gel strength was dependent on the specific NaCl concentration, and the specific reasons need to be further investigated.
2.4 Effect of NaCl concentration and rosemary extract on water retention of gels
As can be seen from Figure 4, when the NaCl concentration was increased from 0.00 mol/L to 0.15 mol/L, the water retention of the MP+rosemary group and the MP group did not change significantly (P＞0.05); as the NaCl concentration continued to increase up to 0.45 mol/L, the water retention of both groups increased significantly (P＜0.05); when the NaCl concentration continued to increase up to 0.60 mol/L, the water retention of the MP group still increased significantly (P＜0.05), while the MP+rosemary group did not increase significantly (P＜0.05), and the MP+rosemary group did not increase significantly (P＜0.05). 0.60 mol/L, the water retention of the MP group still increased significantly (P＜0.05), while the MP+rosemary group did not increase significantly (P＞0.05), and the MP+rosemary group did not increase significantly (P＞0.05). (P＞0.05). There are two necessary conditions for retaining water inside meat: firstly, the meat contains space for water to exist, and secondly, it contains the force to maintain water. It has been demonstrated that the retention of water in meat is mainly dependent on the interactions between water molecules and proteins, such as hydrogen bonding, capillary forces, and dispersive forces[29] .
MP, as a structural protein in meat, provides space for water in the meat; the net negative charge of the protein forms a strong adsorption center, providing a force for water retention. The addition of appropriate amount of NaCl can significantly increase the ionic strength, effectively improve the gelation ability of MP, and make it form a uniform mesh structure. At the same time, with the increase of ionic strength, the net negative charge of MP increased, and the interaction between protein and protein molecules as well as between protein and water was strengthened, which resulted in the increase of the water-holding capacity of the protein in the protein space, and the enhancement of water retention performance[30-31] . Compared with the control group, the water retention of the MP+Rosemary group was significantly higher than that of the MP group at a NaCl concentration of 0.45 mol/L (P<0.05), and the difference was not significant at other NaCl concentrations (P>0.05). Under a certain ionic strength, the addition of a certain amount of rosemary extract could interact with MP and affect the interactions between protein molecules as well as between protein and water, which enhanced the gel formation ability and thus promoted the water holding capacity. In addition, the interaction between phenols and proteins can reduce the gaps between protein polymer chains, thus enhancing the gel structure and water holding capacity [32].
2.5 Effect of NaCl concentration and rosemary extract on rheological properties of MP gels
Rheological properties, as an important characteristic of MP gels, can reflect the quality of the gel. The energy storage modulus (G ' ) reflects the elasticity characteristics of MP gels, and the higher G ' , the stronger the ability to form gels [2]. As can be seen from Figure 5, when the NaCl concentrations were 0.00 and 0.15 mol/L, the G ' of MP+rosemary group and MP group did not show the typical "several" curves, which indicated that the MP failed to form a stable gel structure under these conditions. This is due to the fact that MP was not fully solubilized at low ionic strength, which resulted in weak gel formation and low G'. When the concentrations of NaCl were 0.45 and 0.60 mol/L, the G' gradually increased from 40 ℃ to 46 ℃, and reached the peak at 46 ℃, which was attributed to the beginning of the deconvolution of the α-helix structure of the head of myosin in this range, and the cross-linking of the head of myosin, and the beginning of the formation of the gel; and then, the G' began to decline from 46 ℃, which was attributed to the denaturation of myosin tail, and the stability of the protein was reduced, and the viscosity was reduced; and the G' continued to increase from 50 ℃. From 50 ℃ onwards, G' has been increasing, mainly because most of the myosin molecules may have unfolded into a random coil structure, which enhances the cross-linking between the proteins, thus producing a stable and irreversible gel structure[33-34] . At a certain NaCl concentration, especially at 0.45 mol/L, the G' was higher in the MP+delphinium group than in the MP group without the added extract, which may be attributed to the fact that the added rosemary extract interacted with the MP to create more binding sites and amide bonds, which increased the cross-linking density of the protein and phenol, resulting in the enhancement of the elasticity and viscosity [35-36].
3 CONCLUSIONS
Depending on the specific NaCl concentration (0.45 mol/L), the rosemary extract could effectively "compensate" for the adverse effects of salt reduction on the gel properties of MP. The phenolic acids and terpenes contained in the extract altered the tertiary structure of myosin, and this favorable partial de-helicalization could effectively improve the gel network structure of MP under salt reduction, which was the reason why the gel strength, energy storage modulus, and water retention of MP did not decrease significantly after salt reduction. The results of this study can provide a theoretical basis for the search of new NaCl substitutes.
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