beneficial effects of high-pressure homogenization on the dispersion stability of aqueous hydrolysate from Mytilus edulis
Fengjio Mo, Molin Tu, Fengjio Fn, Cho Wu, Cuiping Yu, Ming Du,∗
aSchool of Food Science and Technology, National Engineering Research Center of Seafood, Dalian Polytechnic University, Dalian 116034, China
bCollege of Food Science and Engineering, Nanjing University of Finance and Economics, Nanjing 210023, China
Owing to the formation of aggregation and gelation during storage, certain proteins and peptides exhibit limited applications in aqueous protein food products. The purpose of this study was to investigate the Influence of homogenization and xanthan gum addition on the dispersion stability of Mytilus edulis hydrolysate (PHM). High-pressure homogenization (HPH) at 360 and 40 bar in the first and second values,respectively, and adding xanthan gum at a concentration of 1 mg/mL showed significantly improvement on the stability of the PHM solution. PHM-xanthan gum solutions (PHMX) showed the highest polypeptide precipitation rate and turbidity retention rate compared with those of PHM. Moreover, the centrifugal precipitation rate of PHMX without HPH was higher than that of homogeneous PHMX. After HPH treatment at 400 bar, the percentage of smaller particles in PHM and PHMX was increased, the aqueous system became more uniform, and the fluorescence intensity reached its maximum. HPH pretreatment improved the polypeptide dispersion stability and turbidity retention rate of PHM and PHMX and reduced the fluorescence intensity. The interactions of xanthan gum and polypeptide render the network microstructure more uniform under the conditions of homogenization, thus improving the dispersion stability of PHMX solutions. Therefore, under the premise of adding xanthan gum, HPH can better enhance the dispersion stability of the polypeptide in PHM.
Homogenized mixed system
Proteins are increasingly being used to promote the development of novel food products, such as sports protein beverages and functional peptide foods . The effective functional properties and nutritional value of proteins can satisfy the various demands of food manufacturers; however, protein dispersion stability limits the shelf life of protein beverage products. The stability of the protein is affected by the inherent protein structure and conformation factors as well as extrinsic elements such as food processing,protein concentration, pH, ionic strength, and temperature . For instance, Cao’s group  has studied that the depolymerization of actin or myosin in duck meat was controlled by ionic strength and processing, such as ultrasound therapy. To better enable the application of protein extracts as functional ingredients in food formulation and prolong their stable storage, such extracts are usually converted into a dry powder form . Mytilus edulis is a typical marine bivalve mollusk dwelling on beach rocks. It is reported containing many types of bioactive components of nutritional value with pharmaceutical activities , for example, nourishing the liver and kidneys, adjusting the blood pressure, curing night sweats,dizziness, etc . Proteins in M. edulis are structurally diversified and comprise a broad range of peptides and all eight essential amino acids . They possess anti-bacterial properties, converting enzyme inhibitory activity as well as anticancer, anti-oxidation, and antihypertensive activity . In the parent protein, these peptides are usually inactive, can be released by hydrolytic enzymes during the hydrolysis processing or gastrointestinal digestion . Accordingly, M. edulis were selected as raw materials for the production of peptide beverages with multifunctional bioactivity.
Generally, certain native proteins rarely show good stability that is desirable for the protein beverage industry . To improve the protein dispersion stability of a beverage, protein structural modifi-cation is often implemented. High-pressure homogenization (HPH)treatment generates the force-induced phenomena of cavitation,shear stress, turbulence, and high hydrostatic pressure when occurring simultaneously, promote the dispersion of aggregates and modify the structure of the protein, and thereby the physicochemical properties of foods [11,12]. Recently, HPH has been proposed for general use in soybean protein and whey protein to modify the structural and functional properties, as this showed some advantages in terms of improved control of character qualities. According to Chen et al. , HPH was reported to accelerate the depolymerization of actin or myosin filaments. However, little information regarding the impact of HPH pretreatment on the polypeptide dispersion stability of M. edulis hydrolysate (PHM) is available.
Xanthan gum is an anionic high-molecular-weight polysaccharide [15,16] that has been globally accepted as a safe food additive for several decades . Xanthan gum is characterized by a secondary structure consisting of a five-fold spiral structure and high molecular weight, which underlies the high viscosity of the solution. The xanthan gum molecule has a cellulosic backbone with coentangled side chains, which protects the backbone and provides excellent dispersion stability over a wide pH range along with tolerance of high salt concentrations. These properties in turn support its wide use in beverages to control component sedimentation.
2. Materials and methods
2.1. Materials and chemicals
M. edulis were purchased from a local commercial shell fish farm (Changxing market, Dalian, China); trypsin (EC 220.127.116.11) was purchased from Solarbio Biological Technology Co., Ltd. (Beijing,China); xanthan gum powder was purchased from Biological Engineering Co., Ltd. (Shanghai, China). All other chemical reagents were of analytical grade and purchased from Sinopharm Group Chemical Reagent Co., Ltd. (Shanghai, China).
2.2. Preparation of PHM
M. edulis was washed thoroughly with deionized water and then the shell and byssus were removed. The muscle was separated manually and pounded to homogenate at 6000 r/min for 5 min in a T25 high-speed dispersion machine (IKA, Staufen, Germany)with a 1:3 (m/m) ratio of muscle to deionized water. The PHM was then pH adjusted to 8.5 using a PB-10 pH meter (Sartorius Stedim Biotech GmbH, Göttingen, Germany) with 0.5 mol/L NaOH,and hydrolyzed by using trypsin (5000 U/g) at 45°C for 2 h. In the process of hydrolysis, the pH was maintained constant by adding 0.2 M NaOH . The reaction was terminated by placing the sample in a boiling water bath for 10 min followed by centrifugation at 8000 × g for 15 min (CR22 N, Merck Hitachi, Hitachi Ltd., Tokyo,Japan). The centrifugation supernatant is diluted with deionized water to a polypeptide concentration of 10 mg/mL, which is PHM.The PHM + HPH is obtained by the twin-stage valve high-pressure homogenizer, where the primary valve pressure is 360 bar and the secondary valve pressure is 40 bar.
2.3. Preparation of PHMX
An appropriate amount of xanthan gum was added to the PHM solution so that the concentration of xanthan gum reached 1 mg/mL. PHMX mixture solution dispersions were obtained upon homogenization in an Ultra-turrax T25 High-Speed Homogenizer(IKA) at 3000 r/min for 3 min. Thereafter, the polypeptide was sufficiently hydrated with the polysaccharide by heating in a water bath kettle (Tianjin Honour Instrument Co., Ltd., Tianjin, China) at 60°C for 30 min . All samples were respectively recirculated three times through a twin-stage valve high-pressure homogenizer(Panda 2 K, GEA Niro Soavi, Parma, Italy) to finally obtain a dispersion having a uniform particle size (PHMX + HPH). HPH pressure was 360 and 40 bar in the first and second valves, respectively .
2.4. Polypeptide concentration determination
The PHM under different treatment conditions were stored for different days by centrifugation with a centrifugal force of 4000 × g for 15 min at 4°C to collect the supernatant for peptide concentration determination. The content of polypeptide in the supernatant was determined using the method of Watters  with minor modification. Specifically, biuret reagent containing 1.5 g cupric sulfate pentahydrate, 6 g sodium tartrate dehydrate, and 300 mL of 10% sodium hydroxide was prepared, then deionized water was added to a volume of 1000 mL to generate the Watters reagent .
2.5. Centrifugal precipitation rate determination
The centrifugal precipitation rate (R) of the samples were measured according to the method of Hu et al.  The samples were transferred to 80 mL centrifuge tubes and centrifuged at 4000 × g for 15 min at 4°C. The supernatant was drained off by inverting the centrifuge tubes and the precipitate was carefully dried with filter paper to remove the residual solution. Centrifuge tube weights alone and with sample or precipitate were accurately determined before and after centrifugation, respectively. The R was calculated as follows:
where Wpis the total centrifugal precipitate plus centrifuge tube weight in grams, Wtis that of the centrifuge tubes alone, and Wsis the total gram weight of the sample plus centrifuge tubes.
2.6. Turbidity retention rate determination
Samples were taken at the same location from the liquid surface and centrifuged (4000 × g, 15 min) to avoid the turbidity difference consequent to the inhomogeneity of the samples. The absorbance at 660 nm was measured  and the turbidity retention rate (T)was calculated according to the following equation:
where CODand SODrepresent the stored centrifugal supernatant and initial state turbidity, respectively.
2.7. Fluorescence spectroscopy observation
The intrinsic fluorescence used for the analysis of structural changes in polypeptides is usually represented by tryptophan (Trp).Samples were diluted 15 times to 3 mL with distilled water and then analyzed using a Hitachi-2700 spectroscopy fluorescence photometer (Hitachi, Tokyo, Japan). The excitation wavelength was 290 nm, and excitation and emission slits were 5 nm. The emission wavelength was detected at the range from 260 nm to 320 nm. The measurements were carried out at 25°C .
2.8. Particle size and zeta potential determination
Particle size and zeta potential of PHM and PHMX solutions were determined using a Zeta sizer 3000 HSa laser particle size analyzer (Malvern Instruments, Worcestershire, UK). The sample was diluted with a phosphate buffer with the ratio of 1:5 to avoid multiple scattering. Zeta potential was measured at 25°C . The intensity distribution (Di) was used to characterize the changes in the average particle size distribution of polypeptides and xanthan gum in PHM and PHMX during storage.
2.9. Scanning electron microscopy observation
The microstructure of PHM and PHMX were observed by SEM(JSM-7800F, Jeol, Tokyo, Japan). At an acceleration voltage of 10 kV, the distribution morphology of polypeptide and xanthan gum particles in the samples were observed in more detail. Prior to observation, the samples were critical point dried and the microparticles were sputter-coated with 2 nm of gold in an argon atmosphere .
2.10. Statistical analysis
This article adopts single factor repeated measures experimental design. Values were subjected to three separate tests under the same conditions with triplicate sample analyses being performed. All data are presented as the means ± standard deviation.To assess the correlation, a Pearson correlation test was performed.The least-squares difference was used to compare mean values among treatments, and significance was established at P < 0.05.ANOVA was conducted and differences between variables were analyzed using the SPSS 12 software package (SPSS Thailand Co.,Ltd., Bangkok, Thailand).
3. Results and discussion
3.1. Effects of HPH on the solubility of polypeptides in PHM
We analyzed the changes of the polypeptide precipitation rate during storage and the physical stability of the PHM (Table 1). The sedimentation of PHM was analyzed by measuring the percentage of total sedimentable solids after centrifugation. As a consequence of particle sedimentation, the higher the value of this parameter,the lower the stability of PHM. Compared with the PHMX system, the peptides in the homogenized PHMX system showed the best dispersion stability after 60 days of storage, and the change in the precipitation rate of the peptide was small. (P < 0.05).Specifically, PHMX storage for 60 days had the highest centrifugal precipitation rate (Fig. 1), whereas the PHM + HPH group showed the lowest rate. Because xanthan gum was added to the PHMX sample, polypeptide and xanthan gum could not form a stable structure without homogenization, which caused a large amount of xanthan gum to precipitate. The turbidity retention rates of homogeneous samples (PHM + HPH and PHMX + HPH) were significantly higher than those of untreated samples (PHM and PHMX), which indicated that HPH pretreatment improves the dispersion stability of the polypeptide system significantly. This result could be attributed to the fact that HPH increased the polypeptide solubility and reduced the particle sizes of polypeptide and/or xanthan gum, resulting in dense and homogeneous gel networks of PHMX following HPH (Table 2). Such compact and uniform microstructure may contribute to the dispersion stability of the system.Similarly, Wu et al.  reported that increased polypeptide solubility and reduced particle size could lead to the improvement of water holding content, which is conducive to system stability.
Table 1Polypeptide precipitation rate during storage of PHM and PHMX. PHM and PHMX at a polypeptide concentration of 10 mg/mL were stored at room temperature after homogenization. The rate of polypeptide precipitation (%) during storage.
Fig. 1. Centrifugal precipitation rate of different storage times. PHM, PHMX and their homogeneous samples were tested separately.
Table 2Untreated and homogenized PHM and PHMX polypeptide solutions were stored to assess turbidity retention over 60 days.
3.2. Effects of HPH on the structural changes of polypeptides
Internal fluorescence is sensitive to changes in the microenvironment around the polypeptides, so changes in fluorescence intensity are believed to be useful for representing the structural change in the polypeptides . Several amino acid residues including Trp, tyrosine (Tyr), and phenylalanine (Phe), can absorb ultraviolet light, which contributes to the absorbance of polypeptides at the wavelength of 270–300 nm. In particular, the position of the chromophore Trp in the molecule is related to the maximum emission wavelength λem . The fluorescence spectra of PHM and PHMX excited at 290 nm are shown in Fig. 2. When excited at 290 nm, PHM and PHMX showed strong fluorescence emission peaks at 295 nm. PHM and PHMX treatment at lower pressures of 400 bar induced a decrease in relative fluorescence intensity,which is in agreement with the results of a previous study showing that following HPH at pressures lower than 1000 bar, the relative fluorescence intensity would be significantly lower than that of the native polypeptide. Molecular rearrangements, energy transfer, ground-state complex formation, collision quenching,and other molecular interactions are likely to cause fluorescence quenching .
For the enzymatic hydrolysate system, the fluorescence intensity of the system with xanthan gum added or homogenized changed significantly (Fig. 2A), which indicates that the polypeptide structure has been modified. The fluorescence intensity of PHM and PHMX samples treated with 400 bar homogenization was considerably lower than that of unhomogenized samples. Shearing, impact, and vibration at lower processing pressures cause the molecular chains to entangle and aggregate, which in turn causes a decrease in fluorescence intensity because a portion of the originally exposed chromophores on the molecular layer becomes trapped within the molecule interior . With the increase of storage days up to 60 days (Fig. 2A–2E), the fluorescence intensity of PHMX continues to increase compared to PHM. Attributed to the addition of xanthan gum without homogenization, the polypeptide molecular chains aggregated by xanthan gum were unfolded and the chromophores were turned outward, which led to more chromophores being exposed at the molecular level . The homogenization uses Dynamic High-pressure Micro fluidization pretreatment. Due to the effects of strong shear and high-frequency vibration, the xanthan gum and polypeptide are better combined,and the exposure of the chromophore is in a stable state. . These results show that homogenization is beneficial for the combination of xanthan gum and polypeptide, and better maintains the stability of the system.
Fig. 2. Fluorescence emission spectra of PHM and PHMX (T =298 K, λex =290 nm). Samples represent 10 mg/mL polypeptide under HPH conditions. Effects of different storage time on the stability of HMP and HMPX prepared by HPH; (A)-(E) represent storage for 0, 7, 14, 30 and 60 days, respectively.
3.3. Effects of HPH on the particle size of PHM
The size distribution was measured by dynamic laser light scattering (suitable for particles ranging from 2 nm to 3000 nm)and the results were expressed as the mean values. Droplet disruption within the homogenate was Influenced by the dispersed phase; particle size was also affected by the dispersed phase during homogenization . Thus, xanthan gum was used as a thickening agent in PHM containing polypeptide to determine the effects of 400 bar HPH and xanthan gum on the particle size. As shown in Fig. 3, the particle size was generally unimodal at the beginning of the storage period, although the PHM had a relatively higher proportion of larger sized particles than the other groups, which indicated a poor dispersion system for the PHM. Upon the addition of xanthan gum, the HPH sample showed better dispersion stability compared with that of PHM. Xanthan gum has an anti-oxidative capacity to increase oxidative stability, while the oxidation can decrease the stability of polypeptides by inducing the aggregation of polypeptides . The proprieties of PHMX such as the particle size and turbidity are expected to be more uniform after homogenization, as xanthan gum can cover a larger interfacial area and consequently prevent the re-agglomeration of the polypeptides.
Fig. 3. Particle size distribution of PHM and PHMX. Figures (A) to (E) show the particle size distribution of PHM and PHMX homogeneity at 0, 7, 14, 30 and 60 days, respectively.
The changes in the size of PHM stored at 25°C for up to 7 days were compared with that of PHMX (Fig. 3B). Both PHM and PHMX showed a gradual decrease in size under HPH conditions. Conversely, the PHMX without HPH remained unchanged, indicating that the aggregation of larger particles leads to instability in the system. Compared with the sample of PHMX, the storage dispersion stability of the PHM and PHMX treated with HPH were shown to be more stable. The particle size of PHMX with xanthan gum was attained at a high level and did not decrease to a substantial degree (Fig. 3C), whereas PHMX with HPH showed bimodal peaks that narrowed upon extension of the storage period (Fig. 3D, 3E). In general, the mixed system without HPH showed precipitation at the bottom of the storage vial especially for the sample with xanthan gum. Increasing the pressure to 175 MPa (1750 bar) could reduce the particle size of lupin-based beverage, but did not change or even started to increase the size at increasing pressure . The authors deduced that HPH treatment above 200 MPa (2000 bar)could induce denaturation of polypeptides, which could interact with other fat globules to forming larger aggregates .
3.4. Effects of HPH on the zeta potential of PHM
Zeta potential is a measure of the strength of repulsion or attraction between particles. Zeta potential is the potential present at this boundary, the intensity of which represents the potential dispersion stability in the mixture system and can be measured by determining the linear velocity of the particles in the electric field.As the particles move, the ions within the boundaries move with them and vice versa . Zeta potential can be used as an index to predict the colloidal dispersion stability, which in turn depends on the interactions of all the component particles. Therefore, zeta potential quantifies the interaction in polypeptide and xanthan gum by predicting stability. The zeta potential is a measure of the repulsive forces between particles. As most colloidal aqueous systems are stabilized by electrostatic repulsion, the greater the repulsion between the particles, the less likely they are to approach and form aggregates, which might result in a more stable mixture system.
Changes in zeta potential during storage are key indicators of colloidal system stability . As shown in Fig. 4, the comparison of the zeta potentials of PHM and PHMX demonstrated a trend toward a decrease of zeta potential; i.e, negative reduction. When PHM and PHMX were stored at 25°C during 0–14 days, the zeta potential increased with storage time (Fig. 4). However, the zeta potential remained unchanged compared with that of homogeneous PHMX upon storage for 60 days. HPH treatment of PHMX had a significant effect on the zeta potential. However, the PHM did not show significant differences compared with the HPH-treated sample,indicating minimal dispersion stability change. Conversely, HPH-treated PHMX samples exhibited significantly better dispersion stability. The results show that the addition of xanthan gum and homogenization can better maintain the stability of the polypeptide. Storage of the PHM and PHMX at 25°C for 14 days resulted in an increase in the values of zeta potential, presumably owing to the ion concentration at the particle interface.
Fig. 4. Zeta potentials of PHM and PHMX. Effects of 40 MPa homogenization pressure on the zeta potential. The average Zeta potential for multiple experiments for the different solutions after 60 days of storage are shown.
Fig. 5. Figures (A) and (B) represents the microstructure of PHM and PHMX and their homogenized samples (PHM + HPH and PHMX + HPH) storage for 0 and 60 days,respectively. Images for storage are shown the scale bar is 50 μm. All pictures were obtained at a magnification of 1000×.
3.5. Effects of HPH on the microstructures of PHM
SEM is widely used to analyze the microstructure of colloids.SEM images of differently treated PHM samples are shown in Fig. 5. Whether it is PHM or PHMX (Fig. 5A, B), the distribution of polypeptides was different, owing to the HPH treatment applied to them. The peptides in the PHM system showed a relatively uniform network structure. After homogenization, the peptide micelles formed a much denser network. Crosslinking and bridging between adjacent micelles were observed. Xanthan gum, after addition, was absorbed, the xanthan gum is adsorbed on the surface of the polypeptide micelle, the network structure was enlarged,and the system become unstable; after homogenization, however, the microstructures of PHM and PHMX were clearer, more uniform and more stable than those of unhomogenized samples.Moreover, the untreated PHMX had many irregular sheet structures, because the size and shape of xanthan gum molecules may affect the microstructures of the polypeptide . HPH can reduce the particle size of the polypeptide or xanthan gum in PHM and PHMX, and make the polypeptide and xanthan gum form a uniform and stable structure during the homogenization process. The microstructure observed in the present study was consistent that reported previously .
In conclusion, HPH could significantly enhance the dispersion stability of PHMX mixtures. Changes in the physical and structural properties of PHM caused by the adsorption of xanthan gum and homogenization could alter the tertiary structure of the polypeptide. Moreover, the results showed that the addition of xanthan gum could enhance the storage dispersion stability of the polypeptide. After HPH treatment, the peptide and xanthan gum showed a comparatively homogeneous network structure as determined by SEM analysis, and the system become more stable.
Declaration of Competing Interest
The authors report no declarations of interest.
This work was supported by the State Key Research and Development Plan¨Modern Food Processing and Food Storage and Transportation Technology and Equipment” (2017YFD0400201)and by the National Natural Science Foundation of China(31771926).
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