Search
Submit Manuscript
3.3CiteScore
Advanced Search
MAP  >  Food Innovation and Advances
2024 Volume 3
Article Contents
Abstract
Introduction
Materials and methods
Materials
Preparation of mixed surimi samples
Rheological characteristics of mixed surimi samples
Apparent viscosity
Dynamic rheological behavior
Strength of mixed surimi gels
Textural properties of mixed surimi gels
Cooking loss of mixed surimi gels
Low-field nuclear magnetic resonance (LF-NMR) of mixed surimi gels
Color difference of mixed surimi gels
Scanning electron microscopy (SEM) of mixed surimi gels
Thiobarbituric acid-reactive substances (TBARS) determination
Statistical analysis
Results and discussion
Rheological properties of surimi gels
Steady-state shear properties
Dynamic elastic properties during gelation
Strength of surimi gels
Textural properties of surimi gels
Cooking loss of mixed surimi gels
Color of surimi gels
Changes in moisture distribution of surimi gels
Scanning electron microscopy of surimi gels
Thiobarbituric acid-reactive substances (TBARS) determination
Conclusions
Author contributions
Data availability
Acknowledgments
Conflict of interest
Supplementary information
Rights and permissions
References
About this article
Next Previous
ARTICLE   Open Access    

Study on physicochemical properties and antioxidant activities of melanoidins extracted from low thermal induced black Lycium barbarum L.

  • College of Food Science and Engineering, Tianjin University of Science and Technology, Tianjin 300457, China

  • Authors contributed equally: Junran Chen, Jie Wang

More Information
  • In this study, static and dynamic desorption methods, infrared spectroscopy and, in vitro antioxidant modeling were used to isolate, purify, and investigate the bioactivity of melanoidins extracted from hypoheat-induced Lycium barbarum L. The results showed that melanoidin fractions with molecular weight in the range of 3−10 kDa were the dominant and most valuable fractions. In the purification phase, the optimal purification conditions were: a loading concentration of 4 mg·mL−1, elution volume of 6 BV, and an elution flow rate of 1 mL·min−1. Purified dominant melanoidin fractions (UF3) exhibited typical Maillard reaction (MR) characteristics in FTIR. The storage stability showed that sunlight and heat treatment exacerbated the instability of the purified UF3. At the same time it was relatively stable under dark conditions and incandescent light, with a retention rate of about 90%. After in vitro digestion, the purified UF3 still exhibited good antioxidant activity, and the DPPH scavenging activity and hydroxyl free radical scavenging ability reached more than 60%.

  • Introduction

    Surimi gel, known as 'concentrated myofibrillar protein'[1], is a kind of gel prepared by processing fish tissue according to fixed steps, such as rinsing, dehydration, and chopping, then adding a certain number of auxiliary materials for crushing, molding, heating and cooling. Salt-soluble myofibrillar protein (mainly myosin) of surimi denatures and unfolds after heating, and then re-crosslinks and polymerizes to form large protein aggregates[2], which is the internal mechanism of forming the gel structure. To enhance the texture and taste of surimi gel products, 2%−3% salt is added to promote the formation of the protein gel network structure and enhance the solubility and functional properties of the products[3,4]. Nonetheless, numerous studies have confirmed that excessive salt intake may result in risks of disease to human health, such as hypertension, and coronary heart disease[5]. Therefore, the development of low-salt surimi products will be widely focused on in future research.

    Recently, two strategies have been innovatively proposed to improve or maintain the gel properties of low-salt surimi products: one is to use exogenous additives or sodium salt substitutes[6−8], and the other is to exploit new processing technologies[9−11]. Glutamine transaminase (TGase) is the most effective surimi quality improver to enhance the functional properties of protein, which could catalyze acyl donors in proteins (γ-Hydroxylamine group) and acyl receptors (lysine residue, primary amine compound, etc.) undergo acyl transfer reaction to form cross-linked structures[12−14]. Previous studies by Jiang et al.[15] revealed that the cross-linking effect of TGase catalysis depends on the content and spatial distribution of available substrates. In addition, lysine, as an ideal exogenous additive, has been widely used in different meat protein gel systems, which can effectively improve the gel properties of low-salt surimi gel[16] and emulsified chicken sausage[17]. Many researchers have spent a lot of time to obtain higher-quality surimi gel products through the combination of two or more exogenous additives. Many scientists devoted themselves to obtaining higher quality surimi gel products through the combination of two or more exogenous additives. For example, Cao et al.[18] demonstrated that lysine (Lys) could bring about the dissociation of actin under the condition of the presence of TGase, promoting the cooking yield and gel properties of oxidative damaged MP. Similar findings reported by Cando et al.[19] indicated that Lys could induce the changes in protein structure, which were favorable terms to heighten the cross-linking effect of TG and improve the strength of surimi gel.

    Recently, the term ε-Poly-lysine (ε-PL) was followed with interest since it's a natural amino acid polymer produced by microbial fermentation, and was speculated that it has a similar molecular structure and potential effect to Lys[20]. ε-PL, which is generally composed of 25−30 lysine residues connected by amide bonds (by ε-Amino and α-Carboxyl group), was often used as an antibacterial agent for the preservation of meat products and aquatic products[21]. Li et al.[21] explored the effect of preservative coating with ε-PL on the quality of sea bass fillets during storage. It turned out that, ε-PL treatment evidently reduced the thiobarbituric acid and volatile base nitrogen values of bass slices during storage, inhibited the growth of microorganisms, and improved the water retention, texture, and flavor characteristics of the fish. A previous study by Cai et al.[20] proved the bacteriostatic and fresh-keeping effect of the composite coating of protein and sodium alginate on Japanese sea bass, and found that the composite coating had an effect on inhibiting the proliferation of sea bass microorganisms (Escherichia coli, lactic acid bacteria, yeast, etc.), fat oxidation, protein degradation, and nucleotide decomposition. As a natural cationic polypeptide, ε-PL has the advantages of no biological toxicity and low viscosity of aqueous solution. It is noteworthy that it can produce strong electrostatic adsorption with negatively charged amino acids[22]. Its effective modification of protein helps it fully interact with gel component molecules and groups[23], which may play a role in enhancing the texture and water retention of protein gel.

    Nevertheless, few reports are based on the addition of ε-PL to explore the influence mechanism on TGase-catalyzed surimi gel properties. The current study sought to shed light on the effects of different additions of ε-PL on TGase-induced cross-linking effect and the performance of composite surimi gel, to provide a theoretical basis and reference for further research and development of 'low salt', efficient and healthy surimi products.

    Materials and methods

    Materials

    Testing materials, including marine surimi, transglutaminase (TG, enzyme activity 100 IU/g), and ε-Polylysine (ε-PL, ≥ 99% purity) were supplied by Jiangsu Yiming Biological Technology Co., Ltd (Jiangsu, China). Egg white protein was purchased from Henan Wanbang Chemical Technology Co., Ltd (Henan, China). All other chemicals were from Shanghai Yuanye Biochemical Co., Ltd (Shanghai, China) and were at least analytical grade. The ingredients of the surimi gel are shown in Table 1.

    Table 1.  Surimi samples with different treatments.
    GroupsSurimi
    (g)    		TGase
    (w/w, %)    		Egg white
    Protein
    (w/w, %)    		ε-PL
    (w/w, %)    		NaCl
    (w/w, %)
    CK300−−−0.5
    TE3000.47.0−0.5
    TE + P13000.47.00.0050.5
    TE + P23000.47.00.010.5
    TE + P33000.47.00.020.5
    TE + P43000.47.00.040.5
    TE + P53000.47.00.060.5
     | Show Table
    DownLoad: CSV

    Preparation of mixed surimi samples

    The prepared block surimi was firstly put into the chopping machine (or tissue masher) and chopped for 2 min, then different mass fractions of ingredients were added, while the chopping time was extended to 5 min (the temperature should be controlled below 10 °C during this process). The exhausted surimi paste was poured into the special mold (50 mm × 20 mm), which was heated in two stages to form the heat-induced surimi gel (40 °C water bath for 40 min; 90 °C water bath for 20 min). Above prepared surimi gel underwent an ice water bath for 30 min and finally stored overnight at 4 °C for further use.

    Rheological characteristics of mixed surimi samples

    Mixed surimi samples with different treatments were determined under a Haake Mars 60 Rheometer (Thermo Fisher Scientific, Germany) with 35 mm stainless steel parallel plates referring to the method described by Cao et al.[18], and each determination was repeated three times. After centrifugation (1,000× g, 3 min) and 4 °C, the degassed surimi sol (~2 g) was equilibrated at 4 °C for 3 min before measurement.

    Apparent viscosity

    The shear stress of the mixed surimi sol was measured at shear rates between 0−100 s−1.

    Dynamic rheological behavior

    The rheological properties of the mixed surimi sols were measured in an oscillatory mode of CD-Auto Strain at 0.02% and 0.1 Hz frequency, respectively. The heating temperature range is set to 20~90 °C while the heating rate is 1 °C·min−1.

    Strength of mixed surimi gels

    The TA-XT Plus physical property analyzer (TA-XT Plus, Stable Micro Systems Ltd, Surrey, UK) was used to analyze the mixed surimi gel strength of each group of samples (n ≥ 3) at room temperature, and the cube-shaped surimi gel samples are measured through the P/0.5 probe. Referring to the method previously described by Fang et al.[24], the test parameters are as follows, pre-test and test rate (1 mm·s−1); rate after measurement (5 mm·s−1); depressing degree (30%); trigger force (5 g); data acquisition rate (400 p/s).

    Textural properties of mixed surimi gels

    Concerning the method of Jirawat et al.[25], texture profile analysis (TPA) was employed to record the hardness, elasticity, cohesion, chewiness, and resilience of mixed surimi gels. It is well known that TPA can explore the textural properties of food through the texture analyzer (TA-XT Plus, Stable Micro Systems Ltd, Surrey, UK) equipped with a P/75 probe to simulate human oral chewing action and obtain the texture characteristic values related to human sensory evaluation. The physical property parameter settings were as follows: downforce (5 g), compression degree (50%), pre-test speed, test speed, and post-test speed (1.0 mm·s−1).

    Cooking loss of mixed surimi gels

    The cooking loss of surimi gel under different treatments was determined referring to the protocol of Dong et al.[26]. The cooked surimi gel sample was instantly absorbed dry and weighed (W5). The cooking loss (CL) is calculated as follows:

    CL(g/100g)=(M1−M2)/M1×100 (1)

    Where M1, weight of the sample before cooking; M2, weight of the sample after cooking.

    Low-field nuclear magnetic resonance (LF-NMR) of mixed surimi gels

    The LF-NMR of the mixed surimi gel was detected by a PQ001-20-025V NMR analyzer (Niumag Analytical Instruments Co., Ltd, Suzhou, China) referring to the previous method conducted by Li et al.[27]. Relevant characteristic parameters were set as follows: sampling frequency (200 KHZ), echo time (0.3 ms), and cumulative number (8). Keeping the surimi gel sample (approximately 2 g) at room temperature for 30 min, they were carefully put into a cylindrical nuclear magnetic tube (15 mm in diameter), and finally, the relaxation time (T2) was recorded using the Carr Purcell Meiboom Gill (CPMG) pulse sequence.

    Color difference of mixed surimi gels

    CM-5 colorimeter (Konica Minolta Sensing, Inc., Tokyo, Japan) was used for surimi gel with different treatments following to the procedure of Wang et al.[28]. Several 1 cm thick slices were cut from surimi samples (n = 3) selected from different treatment groups, and the gel whiteness was calculated according to the following formula:

    Whiteness=100−√(100−L∗)2+a∗2+b∗2 (2)

    Scanning electron microscopy (SEM) of mixed surimi gels

    According to the previously described procedure by Gao et al.[29], the square-shaped surimi gels (4 mm × 4 mm × 4 mm) were immersed in 0.1 mol·L−1 phosphate buffer (pH 7.2) containing 2.5% (v/v) glutaraldehyde for 24 h. The above samples were washed using phosphate buffer (0.1 mol·L−1, pH 7.2) three times and then subsequently dehydrated in a series of alcohol solutions. The microstructures of the mixed surimi gels were imaged using an FEI Verios 460 SEM (FEI Inc., Hillsboro, OR, USA).

    Thiobarbituric acid-reactive substances (TBARS) determination

    The TBARS value was analyzed by the method described by Hu et al.[30] with slight modification. Thiobarbituric acid solution (1.5 mL) and 8.5 mL of trichloroacetic acid solution were added to the sample in turn, the mixture was bathed in water at 100 °C for 30 min. The supernatant was extracted and centrifuged twice (3,000× g, 5 min): (1) The original supernatant (5 mL) was taken and the same amount of chloroform for mixed centrifugation; (2) The second supernatant (3 mL) and petroleum ether (1.5 mL) were taken for mixed centrifugation. Finally, a small amount of lower liquid was taken to determine the absorbance (A532 nm). The final result was expressed in malondialdehyde equivalent (mg·kg−1).

    Statistical analysis

    All statistical analyses of data were investigated by statistical product and service solutions IBM SPSS Statistics version 23.0 (IBM SPSS Inc., Chicago, USA). The LSD all-pairwise multiple comparison method was used for the least significance analysis, and p < 0.05 was considered to indicate significance. The experimental data are expressed as mean ± standard deviation (SD) and plotted using Origin 2019 (Origin Lab, Northampton, MA, USA) software.

    Results and discussion

    Rheological properties of surimi gels

    Steady-state shear properties

    The steady-state shear flow curve can be used to characterize the interaction between proteins. The steady shear flow changes of apparent viscosity (Pa·s) of composite surimi with shear rate (s−1) under different treatments are shown in Fig. 1a. The apparent viscosity of all samples decreased significantly (p < 0.05) with increasing shear rate, exhibiting shear-thinning behavior[31]. In the range of shear rates from 0.1 to 100 s−1, the samples with TGase addition were always higher than the control, indicating that the induction of TGase enhanced the cross-linking of surimi proteins and formed a more stable structure. The surimi samples added with a lower proportion of ε-PL (0.005% and 0.01%) were regarded as lower viscosity fluid (Fig. 1a). The possible reason was that the low concentration of ε-PL provided a weak effect on the pH value of the surimi system. As a consequence, the content of the net charge provided was relatively small and the electrostatic interaction between the surimi proteins was weakened[20].

    Figure 1.  Effect of different treatments on rheological properties of surimi.
    DownLoad: Full-Size Img PowerPoint
    Dynamic elastic properties during gelation

    The elastic modulus (G′) mainly depends on the interaction between protein molecules, which can reflect the change of elasticity in the heat-induced surimi gel. As shown in Fig. 1b, the gel process of surimi in the control group was a thermodynamic process consisting of two typical stages. In the first stage, the value of G' showed a gradual downward trend in the range of 20~50 °C. This is mainly because, (1) the activity of endogenous protease in surimi is increased; (2) the myosin light chain subunits of surimi are dissociated under the action of protease, forming myosin and actin[24]; (3) heat-induced hydrogen bonds break between protein molecules, thus enhancing the fluidity of the gel system[31]. The second stage: From 50 to 73 °C, the G' value increases rapidly. As the temperature continues to rise to 90 °C, the G 'value keeps rising steadily. At this time, the gel network structure gradually became stable and irreversible.

    Compared with several curves in Fig. 1b, TGase treatment could significantly increase the G' value. The change trend of G' value of the samples added with TGase and ε-PL were similar to that of the control group, but the former increased faster and the maximum value of G' was also significantly higher. The temperature corresponding to the first peak of G' gradually decreased (PL1≈PL2 < PL4≈TE < PL3≈PL5 < CK), indicating that the process of protein denaturation and aggregation was advanced. This result showed that the proper amount of ε-PL (0.04%) in combination with TGase can reduce the thermal denaturation temperature of protein-forming gel and improve the forming ability of composite gel, which was unanimous with the changes in texture and properties of surimi gel (Table 2). Furthermore, the addition of a small amount (0.005%) or an excessive amount (0.06%) of ε-PL made the mixed surimi protein system more unstable, affecting the ability of TG-induced gel formation as the last resort[32].

    Table 2.  Effect of different treatments on the textural properties of surimi gels.
    GroupsHardness (g)SpringinessCohesivenessChewiness (g)Resilience
    CK811.40 ± 45.28de0.81 ± 0.01a0.53 ± 0.02b498.36 ± 21.58c0.21 ± 0.01e
    TE1105.80 ± 61.83c0.83 ± 0.01a0.56 ± 0.01b510.40 ± 26.04c0.24 ± 0.00bcd
    TE + P1736.01 ± 18.49e0.80 ± 0.03a0.61 ± 0.00a348.78 ± 18.36d0.23 ± 0.01cde
    TE + P2777.45 ± 39.61de0.80 ± 0.01a0.60 ± 0.02a307.03 ± 12.97e0.22 ± 0.00de
    TE + P3869.66 ± 45.82d0.83 ± 0.02a0.63 ± 0.01a485.06 ± 22.73c0.28 ± 0.00a
    TE + P41492.80 ± 77.13a0.83 ± 0.01a0.60 ± 0.01a721.74 ± 31.55a0.26 ± 0.01ab
    TE + P51314.60 ± 84.10b0.81 ± 0.00a0.60 ± 0.02a652.36 ± 30.24b0.25 ± 0.01bc
    Different lowercase letters in the same column indicated significant differences (p < 0.05).
     | Show Table
    DownLoad: CSV

    Strength of surimi gels

    Gel strength is one of the vital indicators to test the quality of surimi products, which directly affects the texture characteristics and sensory acceptance of the products. The strength of mixed surimi gel with TGase was significantly enhanced by 23.97% (p < 0.05) compared with the control group after heat-inducing. This enhancement is probably caused by the crosslinking promotion of TGase (Fig. 2a). A deeper explanation is that under the catalysis of TGase, the ε-amino group on lysine and the γ-amide group on glutamic acid residues undergo acylation reaction inside or between proteins, forming ε-(γ-Glutamyl)-lysine covalent cross-linking bond which promotes the production of the protein gel network[26].

    Figure 2.  (a) Catalytic reaction of glutamine transaminase and (b) effect of different treatments on gel strength and cooking loss of surimi gel. a−d/A−D: values with different lowercase letters indicate significant difference (p < 0.05).
    DownLoad: Full-Size Img PowerPoint

    On the basis of adding TGase, the strength of the mixed gel with ε-PL concentration ranging from 0.005% to 0.03% was lower than that of TE (Fig. 2b). It was speculated that the surimi sample with low concentration ε-PL was a fluid with lower viscosity, and the interaction between protein and water is strengthened, while the electrostatic interaction between proteins is further weakened[33]. This was also consistent with the change in rheological properties in Fig. 1a. Nevertheless, with the increase of ε-PL content to 0.04%, the gel strength of surimi gel significantly increased and reached the highest value (781.63 g·cm), which was about 21.03% higher than that of the TE group (p < 0.05) (Fig. 2b). The increase of gel strength in this process was obtained due to the following three possible reasons, (1) ε-PL is a cationic amino acid with positive charge, which can improve the pH value of protein or meat product system; (2) The interaction between the ε-amino group of ε-PL and the aromatic residue of protein, namely the cation-π interaction, can change the structure of meat protein[34]; (3) Protein (mixed surimi system) with high ε-amino group content, TGase has strong gel improving ability[35].

    Such synergy (ε-PL = 0.04%), as a comprehensive result of different impactors, is that after the surimi was mixed under the optimal ratio conditions, the synthesis of TGase enzyme catalysis, pH value shift, changes in protein and amino acid composition, etc., increased the strength of various forces, thus forming a denser stereoscopic network structure. Note that when the content of ε-PL reached 0.06%, the gel strength of the corresponding surimi gel decreased by 10% compared with 0.04%. Similarly, others reported the result that alkaline amino acids led to the reduction of the strength of the myosin gel of bighead carp[36].

    Textural properties of surimi gels

    TPA, also known as whole texture, is a comprehensive parameter that determines the sensory quality of surimi gel, including hardness, elasticity, cohesion, chewiness, and resilience[37]. As shown in Table 2, the addition of TGase significantly improved the texture properties of surimi gel (p < 0.05), which is related to TGase's ability to induce surimi protein to form more ε-(γ-Glu)-Lys covalent bond during heating[38] (Fig. 2a). Compared with the cross-linking induced by TGase alone, the gel hardness of the mixed surimi gel treated with ε-PL showed a similar change to the gel strength (Fig. 2b). As the concentration of ε-PL increased from 0.005% to 0.04%, the hardness of surimi gel gradually increased until it reached the maximum (1,492.80 g). At the same time, we also observed that the elasticity, cohesion, and chewiness of the composite gel reached the highest values with the 0.04% ε-PL, and these were higher than those of the TGase-only group. Similar to the findings of Ali et al.[39], the author found that the combination of ε-PL and beetroot extract can effectively replace nitrite, which produced a marked effect in maintaining the color of Frankfurt sausage and improving the texture and performance. However, the data we collated above (Table 2) also showed a clear phenomenon, that is, the addition of ε-PL with lower concentration (0.005%−0.02%) or highest concentration (0.06%) was not conducive to the combination with TGase to improve the texture characteristics of mixed surimi gel.

    Cooking loss of mixed surimi gels

    Cooking loss indicates the water holding capacity of heated surimi, which usually represents the stability of the three-dimensional network structure of surimi gel[40]. The 0.4% TGase significantly reduced the cooking loss of surimi samples (p < 0.05), which was 4.91% lower than that of the control group (Fig. 2b). When the addition of PL (combined with TGase) gradually increased from 0.005% to 0.04%, the cooking loss of surimi showed a decreasing trend and reached the minimum at 0.04%. The ε-PL-added surimi gel samples had higher cooking loss compared with that of only with TGase. Most notably, this may be due to ε-PL is able to further exert the cross-linking effect of TGase. A similar finding was reported in the study by Ma et al.[22], adding ε-PL is able to improve the solubility of myofibrillar proteins. Proteins with high solubility are more easily induced by TGase during heating, which aggravates the cross-linking between ε-PL-protein or protein-protein and forms a disordered gel network structure with relatively weaker water-holding capacity.

    Color of surimi gels

    As shown in Table 3, the high lightness (L*), low yellowness (b*), and high whiteness of surimi in the control group were relatively low, with values of 72.49, 10.42, and 70.56 respectively. After TGase was added to induce cross-linking, these values increased significantly (p < 0.05), which may be related to the photochromic effect of water molecules released from gel matrix under TGase-induced surimi protein cross-linking reported in the previous study[41]. Furthermore, on the basis of adding TGase, it was interesting to see that the L* and whiteness values of surimi gel first increased and then gradually decreased with the increase in ε-PL concentration. The influence of ε-PL combined with TGase on the whiteness of gel can be attributed to three reasons: (1) The addition of ε-PL can effectively increase the substrate content of TGase, and promote the cross-linking between proteins, so as to obtain a much more compact surimi gel structure, which affects the refractive index of light; (2) The water retention performance of the high surimi gel was significantly improved with the addition of ε-PL[42] (Fig. 2b), at this time, the L* value (positively related to the whiteness) value showed a decreasing trend with the decrease of the surface free water content of the gel sample; (3) With the increase of ε-PL concentration, some colored substances, formed due to the accelerated Maillard reaction rate during the preparation of gel, may have an adverse effect on the improvement of gel whiteness[43]. After adding TGase to induce cross-linking, although the whiteness value of composite surimi was significantly enhanced, ε-PL was not enough to further improve the whiteness value of final mixed surimi gel, and high concentration so far as to have a negative impact on the whiteness value.

    Table 3.  Effect of different treatments on the color of surimi gels.
    GroupsL*a*b*Whiteness
    CK72.49 ± 0.82e−0.92 ± 0.01e10.42 ± 0.33d70.56 ± 0.65d
    TE77.82 ± 0.34ab−0.23 ± 0.02c12.29 ± 0.29bc74.64 ± 0.26ab
    TE + P178.39 ± 0.40a−0.14 ± 0.01a12.46 ± 0.12ab75.05 ± 0.41a
    TE + P278.42 ± 0.43a−0.17 ± 0.0b12.46 ± 0.15ab75.07 ± 0.32a
    TE + P377.48 ± 0.16b−0.18 ± 0.02b12.37 ± 0.09abc74.31 ± 0.11b
    TE + P476.34 ± 0.05c−0.22 ± 0.01c12.73 ± 0.02a73.13 ± 0.04c
    TE + P575.48 ± 0.18d−0.26 ± 0.01d12.01 ± 0.14c72.70 ± 0.19c
    Different lowercase letters in the same column indicated significant differences (p < 0.05).
     | Show Table
    DownLoad: CSV

    Changes in moisture distribution of surimi gels

    The water distribution in surimi gel products was measured by LF-NMR[44], and the results were inverted to obtain the transverse relaxation time (T2), which reflects the strength of water fluidity[45]. There are four fitted wave peaks which are assigned to four different water distribution states and wave peak ranges (Fig. 3, Table 4): strong bound water T21 (0.1−1 ms), weak bound water T22 (1−10 ms), non-flowing water T23 (10−100 ms) and free water T24 (100−1,000 ms)[46]. At the same time, similar characteristics (the main peak is centered on T23) are possessed by the water distribution of all surimi gel samples. The addition of TGase increased the proportion of non-flowing water, accompanying the significant decrease in the content of free water (p < 0.05), compared with the control group. It proved that the addition of TGase brought this phenomenon, namely the bound water in surimi gel was partially transferred to the non-flowing, which also indicated the reduction of cooking loss of surimi samples induced by TGase (Fig. 2). On the basis of adding TGase, the low level of ε-PL (0.005%, 0.01% and 0.02%) inhibited the transition of free water in surimi gel to non-flowing water, in which the proportion of T23 decreased by 2.05%, 2.74%, and 1.42% respectively compared with the surimi gel only with TGase, while T24 increased by 15.76%, 14.17%, and 4.43% respectively. The proportion of P23 in the surimi gel sample (ε-PL = 0.04%) was equivalent to that of the TE group, implying that the addition of appropriate ε-PL was not directly connected with the changes in water distribution in surimi after TGase-induced cross-linking, and slight addition would have a negative impact, which was unanimous with the above research results of cooking loss (Fig. 2a).

    Figure 3.  Effect of different treatments on the transverse relaxation time T2 of surimi gel.
    DownLoad: Full-Size Img PowerPoint
    Table 4.  Effect of different treatments on the transverse relaxation time T2 of surimi gel.
    GroupsP21 (%)P22 (%)P23 (%)P24 (%)
    CK2.175 ± 0.186c1.198 ± 0.012e82.863 ± 0.691bc13.764 ± 0.091a
    TE1.938 ± 0.095d2.166 ± 0.043a85.144 ± 1.336a10.752 ± 0.033e
    TE + P12.705 ± 0.067b1.414 ± 0.009d83.435 ± 1.616b12.446 ± 0.105c
    TE + P23.005 ± 0.003a1.845 ± 0.056b82.874 ± 1.739bc12.276 ± 0.108c
    TE + P33.107 ± 0.063a1.715 ± 0.033c83.950 ± 1.587ab11.228 ± 0.086d
    TE + P41.770 ± 0.018d2.099 ± 0.059a85.021 ± 1.756a11.110 ± 0.144d
    TE + P53.121 ± 0.007a1.758 ± 0.107bc82.136 ± 1.091c12.985 ± 0.018b
    Different lowercase letters in the same column indicated significant differences (p < 0.05).
     | Show Table
    DownLoad: CSV

    Scanning electron microscopy of surimi gels

    The effect of different treatment methods on the microstructure of surimi gel was observed by SEM. As shown in Fig. 4, the microstructure of the control surimi gel (Fig. 4a) was observed to be enormously loose, uneven, and irregular, with large pores, which explained the reason why the control surimi gel had low strength and large cooking loss (Fig. 2b). In addition, the morphological properties of another group of surimi gel (TGase-induced) are appreciably different from the scanning electron micrograph mentioned above (Fig. 4b). Numerous compact and evenly distributed small pores were observed, instead of the loose structure in the control sample, which accounted for the fact that TGase could interact with proteins to form a more uniform and orderly three-dimensional network structure in the process of gel formation[47]. Moreover, similar findings were also reported in the previous studies of Dong et al.[26]. When treated with TGase and ε-PL (from 0.05% to 0.04%), the surface of the surimi gel sample gradually became flat through observation (Fig. 4f). We found that some smaller pores were formed and some larger pores were slightly supplemented visually. Remarkably, when the addition of ε-PL increased to 0.06% (Fig. 4g), the structure of surimi gel was not as uniform as before, and some large and irregular holes appeared obviously. This phenomenon might be related to moderate ε-PL, especially 0.02%, which can promote the cross-linking of TGase with protein and the aggregation/denaturation of different components, which agreed with the change of coagulation strength of surimi samples.

    Figure 4.  Effect of different treatments on the microstructure of surimi.
    DownLoad: Full-Size Img PowerPoint

    Thiobarbituric acid-reactive substances (TBARS) determination

    TBARS value, which can reflect the degree of lipid oxidation and rancidity of surimi, is an important indicator for the occurrence of oxidation[48]. Some substances in surimi, such as unsaturated fatty acids, will inevitably undergo oxidative decomposition to produce a large amount of malondialdehyde (MDA), the latter reacts easily with thiobarbituric acid (TBA) reagent to produce red compounds with maximum absorbance at 532 nm. As illustrated in Fig. 5, when TGase and a certain amount of PL (from 0.005% to 0.06%) were added, the TBARs value of surimi gels decreased to 2.03 and 1.67 mg/kg respectively, compared with the control group (2.26 mg/kg). This shows that the addition of both can significantly reduce the TBARS value of surimi gel (p < 0.05), considered to be a cue for the fat oxidation of surimi being inhibited. In addition, we also found that the antioxidant effect of PL was dose-dependent. The ε-PL can be chelated by ferrous ions along with the ability to scavenge free radicals like hydroxyl radicals, ensuring its antioxidant properties[33]. The ε-PL was prospectively considered as an antioxidant additive to prevent surimi protein from deteriorating induced by oxidation[49]. Similarly, making use of lysine or ε-PL as antioxidants in plant protein and meat protein has been widely reported[33, 50]. Fan et al.[42] confirmed that ε-PL addition has a significant effect on maintaining a high total phenolic content and vitamin C levels in fresh lettuce, which also shows that it has certain antioxidant activity.

    Figure 5.  Effect of different treatments on TBARS values of surimi gel. a−f: values with different lowercase letters indicate significant difference (p < 0.05).
    DownLoad: Full-Size Img PowerPoint

    Conclusions

    The addition of ε-PL at different concentrations had an apparent impact on the characteristics of TGase-induced mixed surimi gel, accompanied by the following situations: The 0.04% ε-PL provided a synergistic effect to promote the aggregation and crosslinking of surimi proteins induced by TGase; The rheology, LF-NMR and SEM results showed that the appropriate concentration (0.04%) of ε-PL apparently enhanced the initial apparent viscosity and elasticity of surimi samples, which was conducive to the formation of a more dense and uniform three-dimensional network structure, further limiting the flow of water in surimi and the exudation of hydrophilic substances; Simultaneously, the network strength was strengthened along with the texture properties of the mixed surimi gel. Furthermore, ε-PL had a strong ability to inhibit lipid oxidation in mixed surimi gel, showing a concentration dependence. When the content of ε-PL ran to lowest (0.005%−0.01%) or highest (0.6%), the improvement of the quality of surimi mixed gel was opposite to the above. The results show that ε-PL can be perceived as a prospective polyfunctional food additive to improve the texture properties, nutritional advantages, and product stability of surimi products.

  • The authors confirm contribution to the paper as follows: study conception and design: Cao Y, Xiong YL, Yuan F, Liang G; data collection: Liang G, Cao Y, Li Z, Liu M; analysis and interpretation of results: Li Z, Cao Y, Liang G, Liu Z, Liu M; draft manuscript preparation: Li Z, Cao Y, Liu ZL. All authors reviewed the results and approved the final version of the manuscript.

  • The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

    • Acknowledgments

    This work was financially supported by the Natural Science Basic Research Program of Shaanxi (No. 2023-JC-YB-146), the fund of Cultivation Project of Double First-Class Disciplines of Food Science and Engineering, Beijing Technology & Business University (No. BTBUKF202215), the Innovation Capability Support Plan of Shaanxi (No. 2023WGZJ-YB-27), the Agricultural Technology Research and Development Project of Xi'an Science and Technology Bureau (No. 22NYYF057), and Jiangsu Yiming Biological Technology Co., Ltd. in China.

  • The authors declare that they have no conflict of interest.

  • Supplemental File 1 Screening of macroporous adsorption resin for purification of melanoidins from low thermal induced black Lycium barbarum L.
  • [1]

    Shaheen S, Shorbagi M, Lorenzo JM, Farag MA. 2022. Dissecting dietary melanoidins: formation mechanisms, gut interactions and functional properties. Critical Reviews in Food Science and Nutrition 62(32):8954−71

    doi: 10.1080/10408398.2021.1937509

    CrossRef   Google Scholar

    [2]

    Nooshkam M, Varidi M, Bashash M. 2019. The Maillard reaction products as food-born antioxidant and antibrowning agents in model and real food systems. Food Chemistry 275:644−60

    doi: 10.1016/j.foodchem.2018.09.083

    CrossRef   Google Scholar

    [3]

    Diaz-Morales N, Ortega-Heras M, Diez-Maté AM, Gonzalez-SanJose ML, Muñiz P. 2022. Antimicrobial properties and volatile profile of bread and biscuits melanoidins. Food Chemistry 373:131648

    doi: 10.1016/j.foodchem.2021.131648

    CrossRef   Google Scholar

    [4]

    Rajakaruna S, Pérez-Burillo S, Kramer DL, Rufián-Henares JÁ, Paliy O. 2022. Dietary melanoidins from biscuits and bread crust alter the structure and short-chain fatty acid production of human gut microbiota. Microorganisms 10(7):1268

    doi: 10.3390/microorganisms10071268

    CrossRef   Google Scholar

    [5]

    Suzuki E, Otake S, Hamadate N, Hasumi K. 2020. Kurozu melanoidin, a novel oligoglucan-melanoidin complex from Japanese black vinegar, suppresses adipogenesis in vitro. Journal of Functional Foods 72:104046

    doi: 10.1016/j.jff.2020.104046

    CrossRef   Google Scholar

    [6]

    Kaewtathip T, Wattana-Amorn P, Boonsupthip W, Lorjaroenphon Y, Klinkesorn U. 2022. Maillard reaction products-based encapsulant system formed between chitosan and corn syrup solids: Influence of solution pH on formation kinetic and antioxidant activity. Food Chemistry 393:133329

    doi: 10.1016/j.foodchem.2022.133329

    CrossRef   Google Scholar

    [7]

    Wronkowska M, Zieliński H, Bączek N, Honke J, Topolska J. 2024. Retention of bioactive compounds during technological steps of the production of bread enriched with roasted buckwheat hulls. Food Chemistry 460:140645

    doi: 10.1016/j.foodchem.2024.140645

    CrossRef   Google Scholar

    [8]

    Böhm W, Zinke L, Rehle AK, Henle T. 2023. Role of proteins in the formation of melanoidins during coffee roasting. Journal of Agricultural and Food Chemistry 71(47):18499−509

    doi: 10.1021/acs.jafc.3c05425

    CrossRef   Google Scholar

    [9]

    Makiso MU, Tola YB, Ogah O, Endale FL. 2023. Bioactive compounds in coffee and their role in lowering the risk of major public health consequences: a review. Food Science & Nutrition 12(2):734−64

    doi: 10.1002/fsn3.3848

    CrossRef   Google Scholar

    [10]

    Ma RH, Zhang XX, Ni ZJ, Thakur K, Wang W, et al. 2023. Lycium barbarum (goji) as functional food: a review of its nutrition, phytochemical structure, biological features, and food industry prospects. Critical Reviews in Food Science and Nutrition 20:10621−35

    doi: 10.1080/10408398.2022.2078788

    CrossRef   Google Scholar

    [11]

    Wang JJ, Zhang WW, Guan ZJ, Thakur K, Hu F, et al. 2023. Effect of fermentation methods on the quality and in vitro antioxidant properties of Lycium barbarum and Polygonatum cyrtonema compound wine. Food Chemistry 409:135277

    doi: 10.1016/j.foodchem.2022.135277

    CrossRef   Google Scholar

    [12]

    Ding X, Zhang Y, Li J, Yan S. 2024. Structure, spectral properties and antioxidant activity of melanoidins extracted from high temperature sterilized lotus rhizome juice. International Journal of Biological Macromolecules 270:132171

    doi: 10.1016/j.ijbiomac.2024.132171

    CrossRef   Google Scholar

    [13]

    Zhu Z, Zhang Y, Wang W, Huang Z, Wang J, et al. 2021. Structural characterisation and antioxidant activity of melanoidins from high-temperature fermented apple. International Journal of Food Science & Technology 56(5):2471−80

    doi: 10.1111/ijfs.14881

    CrossRef   Google Scholar

    [14]

    Al-Shehri SA. 2021. Efficacy of black garlic extract on anti-tumor and anti-oxidant activity enhancement in rats. Clinical Nutrition Open Science 36:126−139

    doi: 10.1016/j.nutos.2021.03.005

    CrossRef   Google Scholar

    [15]

    Matsuse K, Hirata S, Abdelrahman M, Nakajima T, Iuchi Y, et al. 2024. Comparative Studies of Bioactivities and Chemical Components in Fresh and Black Garlics. Molecules 29(10):2258

    doi: 10.3390/molecules29102258

    CrossRef   Google Scholar

    [16]

    Liu R, Wu Q, Xu J, Gao Y, Zhi Z, et al. 2023. Isolation of melanoidins from heat-moisture treated ginseng and its inhibitory effect on choline metabolism. Journal of Functional Foods 100:105370

    doi: 10.1016/j.jff.2022.105370

    CrossRef   Google Scholar

    [17]

    Tores de la Cruz, S, Iriondo-DeHond A, Herrera T, Lopez-Tofiño Y, Galvez-Robleño C, et al. 2019. An assessment of the bioactivity of coffee silverskin melanoidins. Foods 8(2):68

    doi: 10.3390/foods8020068

    CrossRef   Google Scholar

    [18]

    Zhang Q, Chen M, Emilia Coldea T, Yang H, Zhao H. 2023. Structure, chemical stability and antioxidant activity of melanoidins extracted from dark beer by acetone precipitation and macroporous resin adsorption. Food Research International 164:112045

    doi: 10.1016/j.foodres.2022.112045

    CrossRef   Google Scholar

    [19]

    Starowicz M, Ostaszyk A, Zieliński H. 2021. The relationship between the browning index, total phenolics, color, and antioxidant activity of Polish-Originated honey samples. Foods 10(5):967

    doi: 10.3390/foods10050967

    CrossRef   Google Scholar

    [20]

    Yang S, Fan W, Nie Y, Xu Y. 2023. The formation and structural characteristics of melanoidins from fermenting and distilled grains of Chinese liquor (baijiu). Food Chemistry 410:135372

    doi: 10.1016/j.foodchem.2022.135372

    CrossRef   Google Scholar

    [21]

    Wu J, Jin Y, Zhang M. 2021. Evaluation on the physicochemical and digestive properties of melanoidin from black garlic and their antioxidant activities in vitro. Food Chemistry 340:127934

    doi: 10.1016/j.foodchem.2020.127934

    CrossRef   Google Scholar

    [22]

    Oracz J, Nebesny E. 2019. Effect of roasting parameters on the physicochemical characteristics of high-molecular-weight Maillard reaction products isolated from cocoa beans of different Theobroma cacao L. groups. European Food Research and Technology 245(1):111−28

    doi: 10.1007/s00217-018-3144-y

    CrossRef   Google Scholar

    [23]

    Kim JS. 2020. Antioxidant activity of various soluble melanoidins isolated from black garlic after different thermal processing steps. Preventive Nutrition and Food Science 25(3):301−9

    doi: 10.3746/pnf.2020.25.3.301

    CrossRef   Google Scholar

    [24]

    Zhao Y, Ding Y, Wang D, Deng Y, Zhao Y. 2021. Effect of high hydrostatic pressure conditions on the composition, morphology, rheology, thermal behavior, color and stability of black garlic melanoidin. Food Chemistry 337:127790

    doi: 10.1016/j.foodchem.2020.127790

    CrossRef   Google Scholar

    [25]

    Peña-Correa RF, Wang Z, Mesa V, Ataç Mogol B, Martínez-Galán JP, et al. 2023. Digestion and gut-microbiota fermentation of cocoa melanoidins: an in vitro study. Journal of Functional Foods 109:105814

    doi: 10.1016/j.jff.2023.105814

    CrossRef   Google Scholar

    [26]

    Affes S, Nasri R, Li S, Thami T, Van Der Lee A, et al. 2021. Effect of glucose-induced Maillard reaction on physical, structural and antioxidant properties of chitosan derivatives-based films. Carbohydrate Polymers 255:117341

    doi: 10.1016/j.carbpol.2020.117341

    CrossRef   Google Scholar

    [27]

    Liu Z, Wang C, Chen H, Ren X, Li W, et al. 2022. Effect of changing the melanoidins by decoction on the release of volatiles in Zhenjiang aromatic vinegar. Food Research International 158:111453

    doi: 10.1016/j.foodres.2022.111453

    CrossRef   Google Scholar

    [28]

    Yang H, Zhang Q, Zeng Y, Cheng C, Coldea TE, et al. 2024. Differences in structure, stability and antioxidant activity of melanoidins from lager and ale beers. LWT 205:116517

    doi: 10.1016/j.lwt.2024.116517

    CrossRef   Google Scholar

    [29]

    Naibaho J, Korzeniowska M. 2021. Brewers' spent grain in food systems: processing and final products quality as a function of fiber modification treatment. Journal of Food Science 86:1532−51

    doi: 10.1111/1750-3841.15714

    CrossRef   Google Scholar

    [30]

    Patrignani M, Rinaldi GJ, Rufián-Henares JÁ, Lupano CE. 2019. Antioxidant capacity of Maillard reaction products in the digestive tract: an in vitro and in vivo study. Food Chemistry 276:443−50

    doi: 10.1016/j.foodchem.2018.10.055

    CrossRef   Google Scholar

    [31]

    Oracz J, Zyzelewicz D. 2019. In vitro Antioxidant activity and FTIR characterization of high-molecular weight melanoidin fractions from different types of cocoa beans. Antioxidants 8:560

    doi: 10.3390/antiox8110560

    CrossRef   Google Scholar

    [32]

    Oracz J, Lewandowska U, Owczarek K, Caban M, Rosicka-Kaczmarek J, et al. 2024. Isolation, structural characterization and biological activity evaluation of melanoidins from thermally processed cocoa beans, carob kibbles and acorns as potential cytotoxic agents. Food Chemistry 442:138423

    doi: 10.1016/j.foodchem.2024.138423

    CrossRef   Google Scholar

    [33]

    Hussain A, Hussain M, Ashraf W, Karim A, Muhammad Aqeel S, et al. 2024. Preparation, characterization and functional evaluation of soy protein isolate-peach gum conjugates prepared by wet heating Maillard reaction. Food Research International 192:114681

    doi: 10.1016/j.foodres.2024.114681

    CrossRef   Google Scholar

    [34]

    Nooshkam M, Varidi M. 2020. Whey protein isolate-low acyl gellan gum Maillard-based conjugates with tailored technological functionality and antioxidant activity. International Dairy Journal 109:104783

    doi: 10.1016/j.idairyj.2020.104783

    CrossRef   Google Scholar

    [35]

    Mohsin GF, Schmitt FJ, Kanzler C, Epping JD, Buhrke D, et al. 2020. Melanoidin formed from fructosylalanine contains more alanine than melanoidin formed from D-glucose with L-alanine. Food Chemistry 305:125459

    doi: 10.1016/j.foodchem.2019.125459

    CrossRef   Google Scholar

    [36]

    Farag MA, von Bergen M, Saleh BM, Homsi MN, Abd El-Al MS. 2021. How do green and black coffee brews and bioactive interaction with gut microbiome affect its health outcomes? Mining evidence from mechanistic studies, metagenomics and clinical trials. Trends in Food Science & Technology 118:920−37

    doi: 10.1016/j.jpgs.2021.11.004

    CrossRef   Google Scholar

  • Cite this article

    Chen J, Wang J, Liu Y, Li H, Wang W, et al. 2024. Study on physicochemical properties and antioxidant activities of melanoidins extracted from low thermal induced black Lycium barbarum L. Food Innovation and Advances 3(3): 288−294 doi: 10.48130/fia-0024-0027
    Chen J, Wang J, Liu Y, Li H, Wang W, et al. 2024. Study on physicochemical properties and antioxidant activities of melanoidins extracted from low thermal induced black Lycium barbarum L. Food Innovation and Advances 3(3): 288−294 doi: 10.48130/fia-0024-0027
    shu

Figures(6)  /  Tables(1)

Download PDF

Article Metrics

Article views(2088) PDF downloads(316)

Other Articles By Authors

ARTICLE   Open Access    

Study on physicochemical properties and antioxidant activities of melanoidins extracted from low thermal induced black Lycium barbarum L.

  • College of Food Science and Engineering, Tianjin University of Science and Technology, Tianjin 300457, China

  • Received: 30 March 2024
  • Revised: 11 August 2024
  • Accepted: 12 August 2024
  • Published online: 30 August 2024
Food Innovation and Advances  3,  2024, 3(3): 288−294  |  Cite this article

Abstract: In this study, static and dynamic desorption methods, infrared spectroscopy and, in vitro antioxidant modeling were used to isolate, purify, and investigate the bioactivity of melanoidins extracted from hypoheat-induced Lycium barbarum L. The results showed that melanoidin fractions with molecular weight in the range of 3−10 kDa were the dominant and most valuable fractions. In the purification phase, the optimal purification conditions were: a loading concentration of 4 mg·mL−1, elution volume of 6 BV, and an elution flow rate of 1 mL·min−1. Purified dominant melanoidin fractions (UF3) exhibited typical Maillard reaction (MR) characteristics in FTIR. The storage stability showed that sunlight and heat treatment exacerbated the instability of the purified UF3. At the same time it was relatively stable under dark conditions and incandescent light, with a retention rate of about 90%. After in vitro digestion, the purified UF3 still exhibited good antioxidant activity, and the DPPH scavenging activity and hydroxyl free radical scavenging ability reached more than 60%.

    HTML

    Introduction

    • The Maillard reaction (MR) is known as non-enzymatic browning, which occurs widely during the heating process of food. Melanoidins, the product of the MR, are formed by the condensation of reducing sugar carbonyl and amino group[1]. Melanoidins have been shown to improve food quality, such as color, flavor, and biological activity[2]. For example, melanoidins extracted from bread and biscuits exhibit antimicrobial effects on certain molds and yeasts, which could improve the shelf life and safety of food as a natural antimicrobial agent; melanoidins extracted from bread could serve as a carbon source for Bifidobacterium. Melanoidins from black vinegar significantly suppressed adipogenesis of 3T3-L1[3−5]. Currently, there are many reports on the antioxidant capacity of melanoidins. It is generally recognized that melanoidins have excellent antioxidant activities[6−9]. These excellent properties demonstrate melanoidins have considerable application value in the food industry, which can be used as novel food ingredients.

      Lycium barbarum L. has been used as a traditional food and herbal medicine in China, as described in the Compendium of Materia Medica. A large number of bioactive substances, including polysaccharides, phenols, proteins, alkaloids, and vitamins, have been found in Lycium barbarum L.[10,11]. Importantly, the bioactivity of Lycium barbarum L. can be significantly enhanced by the concoction process. It has been reported that this increase in activity can be attributed to the production of melanoidin[12]. Black garlic and black apple, which have been extensively studied, also belong to this category of food products and the presence of melanoidins in the system also increases the functional properties of the products[13−15]. However, the development of novel products with melanoidin from Lycium barbarum L. as a raw material by heat-high humidity or low-temperature-induced processing has not been intensively investigated; in particular, it was carried out with the Ningxia wolfberry as the selective target. Therefore, melanin was extracted and purified from Lycium barbarum L. and a preliminary study was conducted on its physicochemical properties, digestive properties, and functional activities. This study will provide theoretical support for further research on the application of melanoidins extracted from Lycium barbarum L. as functional foods or dietary additives, and provide a new avenue for the utilisation of Lycium barbarum L.

    Materials and methods

      Materials and preparation of heat-induced black Lycium barbarum

    • Dried Lycium barbarum L. with 20% water content was provided by Zaokang Wolfberry Incorporated Company (Ningxia Province, China). Dried Lycium barbarum L. was placed in a constant temperature and humidity chamber, then treated for 48 h at 60 °C and 50% humidity to obtain black Lycium barbarum L. (BLB). Pepsin (10,000 U/g) was purchased from Hefei Qiansheng Biotechnology Co., Ltd. ( Anhui Province, China). Trypsin (250 U/mg) and α-amylase (10,000 U/g) were purchased from Jiangsu Ruiyang Biotechnology Co., Ltd. (Jiangsu Province, China).

      Preparation of melanoidins

    • Melanoidins were extracted according to the method described by Liu et al.[16] with some modifications, as described in the Supplemental File 1. BLB powder (20 g) was extracted with 200 mL of 30% ethanol solution (v/v) in a water bath at 45 °C for 90 min and then filtered to collect the supernatant. The collected supernatant was added to an anhydrous alcohol solution to achieve an ethanol concentration of 70% and then allowed to stand for 12 h at 4 °C to precipitate. Then the supernatant was filtered with filter paper. The filtrate was dialyzed for 48 h to remove compounds with a molecular weight (MW) below 1 kDa and then freeze-dried. Finally, the crude melanoidins were extracted and packed in vacuum-sealed bags for further use.

      Isolation and evaluation of melanoidin fractions

    • The isolation of crude melanoidins was conducted using a method previously reported by Tores de la Cruz et al.[17] with some modifications. The prepared crude melanoidin solution was sequentially ultrafiltered three times using ultrafiltration membranes with molecular weight cut-off (MWC) of 50, 10, and 3 kDa to separate the fractions UF1 (ultrafiltration fraction 1, MWC > 50 kDa)，UF2 (10 kDa < MWC < 50 kDa)，UF3 (3 kDa < MWC < 10 kDa) and UF4 (< 3 kDa) respectively. The ultrafiltration procedure was repeated three times to collect the four melanoidin fractions. The fractions were freeze-dried, weighed and then stored at −20 °C. The chemical composition and antioxidant activity were analyzed to preliminarily evaluate the properties of the four fractions.

      Orthogonal experimental design of resin purification

    • According to the preliminary analysis of four fractions, the target fraction was confirmed. The target fraction was further purified according to the method proposed by Zhang et al.[18] with some modifications. Briefly, dynamic adsorption and desorption tests were performed with the pretreated AB-8 resin. Twenty grams of the pretreated AB-8 resin was loaded into the glass column and a sample of the target fraction solution was loaded, and then eluted with 40% ethanol. First, three variables (loading concentration, elution volume, and elution flow rate) that could influence the yield of melanoidin purification were evaluated in single factor experiment. The conditions for the single factor experiment were set as follows: Loading concentrations (2, 4, 6, 8, and 10 mg·mL−1), elution volume (4, 5, 6, 7, and 8 BV) and elution flow rate (1, 2, 3, 4, and 5 mL·min−1). Based on the results of the single factor experiments, an orthogonal L9 (33) test (as shown in Table 1) was performed. The purified melanoidin obtained under the optimal conditions was then analyzed for structure, stability, and antioxidant activity.

      Table 1.  Analysis and results of orthogonal L9 (33) experimental design.

      Number Factors Results
      a: Loading concentration (mg·mL−1) b: Elution volume (BV) c: Elution flow rate (mL·min−1) Yield (%)
      1 6 6 1 0.487
      2 6 7 2 0.332
      3 6 8 3 0.243
      4 8 6 2 0.398
      5 8 7 3 0.265
      6 8 8 1 0.255
      7 10 6 3 0.253
      8 10 7 1 0.348
      9 10 8 2 0.273
      k1 0.351 0.376 0.360
      k2 0.306 0.315 0.334
      k3 0.291 0.257 0.253
      R 0.0594 0.119 0.107
      Order of importance b > c > a
      Optimal level a1b1c1

      Determination of the chemical constituents of melanoidin fractions

    • The contents of reducing sugar and total sugar were determined by the DNS method and phenol sulfate colorimetry method, respectively. The total phenolic content was determined using the Folin-Ciocalteu method[19]. The protein content was carried out using the Bradford method using bovine serum albumen as standard[20]. Using rutin as the standard sample, the total flavonoid content was determined. A 0.5 mL sample was mixed with 0.4 mL of 5% NaNO2 and then incubated for 6 min. Afterward, 0.4 mL of 10% Al(NO3)3 was added and reacted for 6 min. Finally, 4 mL of 4% NaOH and 4.7 mL of 70% ethanol were added to the reacted solution. Then the sample solution was incubated for 10 min and the absorbance was determined at 505 nm.

      Antioxidant activity determination

      Hydroxyl radical scavenging activity

    • The hydroxyl radical scavenging activity was determined using the method described by Wu et al.[21] with some modifcations. Briefly, 1 mL of FeSO4 solution (5 mmol·L−1), 4 mL of salicylic acid solution (5 mol·L−1) and 3 mL of distilled water were mixed with 1 mL sample. Then, 1 mL of H2O2 (5 mmol·L−1) was added and reacted at 37 °C for 10 min. The absorbance of the reacted solution was measured at 510 nm, and the hydroxyl radical scavenging activity was calculated using the following equation:

      Hydroxylradicalscavengingability(%)=A0−(A1−A2)A0×100% (1)

      where, A0: the absorbance of the blank; A1: the absorbance of the control; A2: the absorbance of the sample.

      Ferric-reducing antioxidant power

    • The antioxidant power was measured according to the method described by Oracz & Nebesny[22]. One mL of sample (0.5 mg·mL−1) was mixed with 2.5 mL of sodium phosphate buffer (pH = 6.6, 0.2 mol·L−1) and 2.5 mL of potassium ferricyanide (1%, v/v). The mixed solution was incubated in a water bath at 50 °C for 20 min. 2.5 mL of 10% trichloroacetic acid was added when the solution was cooled to room temperature. One mL of ferric chloride (1%, v/v) and 2.5 mL of distilled water were added into 2.5 mL of this mixture. Finally, the absorbance of the mixture was determined at 700 nm after incubation for 10 min.

      DPPH radical scavenging activity

    • The DPPH radical scavenging activity of melanoidin was evaluated following previous reports by Kim[23] with slight modifications. Four mL of 95% ethanol solution and DPPH-ethanol mixed solution (2 mL, 0.05 mg·mL−1) were added to 1 mL of sample solution and then allowed to react for 30 min under light protection. The absorbance value of the solution was determined at 517 nm. The DPPH radical scavenging activity (%) was calculated as follows:

      DPPHradicalscavengingactivity(%)=A0−(A1−A2)A0×100% (2)

      where, A0: the absorbance of the blank; A1: the absorbance of the control; A2: the absorbance of the sample.

      Fourier transform infrared spectroscopy (FTIR)

    • The sample obtained above (1 mg) was mixed with dried KBr (100 mg) and pressed into a thin sheet with a tablet press after being ground. Then the sample was analyzed using a spectrometer in the 400−4,000 cm−1 range.

      Stability assay

    • The stability of the purified melanoidins was examined according to a previous study[24]. Briefly, the stability was estimated by exposing the samples to different heating temperatures (40, 50, 60, 70, and 80 °C) and different light conditions (sunlight, incandescent light, darkness). The absorbance of the samples exposed to different heating temperatures was determined every 1 h at 420 nm. And the absorbance of the samples exposed to different light conditions were determined every 1 d. The retention rate (R) was calculated using the following equation:

      R=A1−A0A0×100% (3)

      where, R: Retention rate; A0: The absorbance value of the initial sample; A1: The absorbance value of the sample after treatment under different conditions.

      Antioxidant activity in vitro simulated digestion

    • The purified melanoidins were digested according to the method described by Peña-Correa et al.[25]. Briefly, in the oral digestion phase, 1.0 g of sample and 20 mL of simulated salivary fluid (pH adjusted to 7.0) were mixed and incubated at 37 °C for 2 min. In the gastric digestion phase, the sample after oral digestion was mixed with 10 mL of simulated gastric fluid (pH adjusted to 1.2) and incubated at 37 °C for 2 h. In the intestinal digestion phase, 10 mL of simulated intestinal fluid (pH adjusted to 7.0) was added to the above gastric digestion mixture and incubated at 37 °C for 2 h. The samples before and after each digestion step were collected and freeze-dried for further analysis of antioxidant activity. Antioxidant activity was analyzed according to the method described above.

      Statistic analysis

    • All experimental results were determined in triplicate and the data were expressed as mean ± standard deviation (Mean ± SD). ANOVA accompanied and Duncan’s multiple range tests were performed to examine statistical differences across groups using SPSS software (version 19.0 for Windows, SPSS Inc., Chicago, IL, USA).

    Results and discussion

      Isolation and preliminary evaluation of melanoidin fractions

      Content of isolated melanoidin fractions

    • Four melanoidin fractions with different MW were obtained by ultrafiltration. As shown in Fig 1a, the melanoidin fraction with HMW presented a deeper brown color than in the LMW fractions. The results indicate that the HMW melanoidins were stronger colorants that contribute more to the color of thermally treated foods[21,26]. As shown in Fig. 1b, UF3 showed the highest content (59.37% ± 3.66%), followed by UF4 (24.21% ± 3.01%), UF2 (12.90% ± 1.25%) and UF1 (3.52% ± 0.98%), indicating that melanoidin fractions with LMW were dominant fractions. This result could be due to the low temperature of the reaction system, which leads to the formation of a large amount of LMW melanoidin in the early phase of MR. Melanoidin is the product of the Maillard reaction between proteins and sugars. It has been shown that the reaction time and temperature are related to the size of the molecular weight. When the reaction temperature is low, a large amount of LMW melanoidin is formed in the early phase of MR. LMW melanoidin can be used as a reaction intermediate with other Maillard reaction products to polymerize or crosslink to form HMW melanoidin in the later stage of the reaction with the extension of heating time[27,28]. This finding is consistent with the fact that the molecular weight of melanoidin in ale beers and lager beers increased with the roasting time of the ingredients and the intensity of the heating[28].

      Figure 1. 

      The physicochemical properties of melanoidin fractions with different MW extracted from induced black Lycium bararum L., including (a) color, (b) proportion of ultrafiltration fraction, and (c) chemical composition. UF1 (Ultrafiltration fraction 1, MWC > 50 kDa), UF2 (10 kDa < MWC < 50 kDa), UF3 (3 kDa < MWC < 10 kDa) and UF4 (< 3 kDa).

      Chemical compositions of isolated melanoidin fractions

    • Chemical compositions commonly retained in melanoidins by ultrafiltration membranes include total sugar, reducing sugar, total phenol, and protein[21]. The content of chemical compositions in four melanoidin fractions are shown in Fig. 1c. The total flavonoid, which is abundant in Lycium barbarum L., was not detected in the four melanoidin fractions. The total phenol exhibited a low content of less than 0.5% in four melanoidin fractions. The total phenol content increased with the increase in MW. The findings were similar to those of Naibaho & Korzeniowska[29], who observed that melanoidins in Brewers' spent grain with the higher molecular weight contained more total polyphenol. The reducing sugar and protein also presented low content indicating that the reducing sugar was fully reacted with protein in the Maillard reaction. The total sugar in four melanoidin fractions showed the highest content in the range of 5.91% to 7.14%. And a significant difference in the total sugar content was observed between UF4 and other melanoidin fractions. This is probably the result of the ultrafiltration membrane with MWC < 3 kDa efficiently removing the unreacted high molecular weight sugars.

      Antioxidant activities of isolated melanoidin fractions

    • As shown in Fig. 2a, the reducing power of all melanoidin fractions increased with the increase of sample concentration. The reducing power of melanoidin fractions with LMW was found to be significantly higher than those of melanoidin fractions with HMW. The reducing power of UF4 in concentration of 2.0% was about 0.46 times higher than that of UF1. The scavenging activities of four melanoidin fractions with different concentrations against the hydroxyl radical are shown in Fig. 2b. The scavenging activity against hydroxyl radicals of the four melanoidin fractions was positively associated with the concentration. The scavenging activity was enhanced significantly with the decreasing of MW. The scavenging activity against hydroxyl radicals of UF4 at a concentration of 2.0% was 18% higher than that of UF1. The results consistent with a previous report[28] indicated that melanoidin with LMW prepared from beers showed stronger antioxidant activity. It can be seen from Fig. 2c, a positive correlation between the concentrations and radicals scavenging activities of all melanoidin fractions was also observed. The DPPH radicals scavenging activity of UF2 and UF3 was significantly stronger than that of UF1 and UF4. The reason for this result may be that the ability of melanin to scavenge DPPH free radicals is expressed by the donation of hydrogen atoms and UF2 and UF3 may contain more hydrogen to form stable DPPH-H molecules[13,30].

      Figure 2. 

      The antioxidant activities of melanoidin fractions with different MW, including (a) reducing power, (b) hydroxyl radical scavenging activity, and (c) DPPH radical scavenging activity.

      Optimization of UF3 purification conditions

    • According to the above analysis of four fractions, UF3 was confirmed as the target fraction for further research and application. Seen from the results of single-factor experiments presented in Fig. 3a−c, the highest yield of melanoidins were 33.5%, 38.5%, and 43.5% at the loading concentration of 8 mg·mL−1, the elution volume of 7 BV and the elution flow rate of 2 mL·min−1 respectively. Based on the single factor experiments, orthogonal experiments with these three variables were performed. Table 1 presents the optimization results. Among these factors that influenced the yield of melanoidins, the elution volume (b) was the most significant influencing factor, followed by the elution flow rate (c), and the loading concentration (a). The optimum parameters for UF3 purification were a1b1c1. The highest yield of UF3 was obtained when the loading concentration, the elution volume, and the elution flow rate were 4 mg·mL−1, 6 BV, and 1 mL·min−1 , respectively.

      Figure 3. 

      Impact of (a) loading concentration, (b) elution volume, and (c) elution flow rate on the purification yields.

      FTIR analysis of purified UF3

    • The FTIR spectra of the purified UF3 are shown in Fig. 4. An absorption band at 3,394.98 cm−1 was observed. The absorption bands at 3,600−3,200 cm−1 is assigned to stretching vibration of the hydroxyl group (O-H) or amide group (N-H)[29]. The signal peak presented at 2,923.63 cm−1 could be attributed to the stretching vibration of C-H. The C-H (CH3 and CH2) band are found around 2,943 cm−1[31]. In previous studies, the melanoidin skeleton is rich in O-H, N-H, and -CH functional groups[31−33]. The absorption bands of amide were observed at 1,639.25, 1,404.61, and 1,242.00 cm−1, which was attributed to C=O stretching and C=N stretching modes (amide I, 1,700−1,600 cm−1), N-H bending and C-N stretching modes (amide III, 1,450−1,240 cm−1), respectively[34]. Two absorption peaks at 1,075.28 and 1,038.23 cm−1 can be assigned to C-O-C bending. In addition, a band was observed at 581.29 cm−1 ascribed to the skeletal modes of pyranose rings[35]. In summary, the structure of purified UF3 was close to the typical melanoidins structure, indicating that the UF3 was effectively prepared.

      Figure 4. 

      FT-IR spectra of purified UF3.

      Stability of purified UF3

    • The stability of purified UF3 under different light conditions and temperatures was investigated. Under different light conditions, the retention rate of all melanoidin samples showed a downward trend with increasing exposure time (Fig. 5a). The downward trend of melanoidin samples exposed to sunlight showed a greater decrease than the other two groups. The retention rate of the melanoidin sample was reduced by about 17.23% after 10 d of sunlight exposure. It could be that the ultraviolet rays contained in sunlight accelerated the oxidative degradation of melanoidin. Similarly, melanoidin from black garlic also showed a decreasing trend under ultraviolet conditions[24]. The retention rate of melanoidin treated with incandescent light was similar to that of dark conditions with a retention rate of about 90%, suggesting that the purified UF3 can be stored and used under incandescent light. As shown in Fig. 5b, the retention rate of the melanoidin samples treated with different heating temperatures showed a fluctuating trend, indicating that the heat treatment causes heat polymerization and heat degradation of the purified UF3[28]. Therefore, heat treatment could aggravate the thermal instability of the melanoidin.

      Figure 5. 

      Stability results of purified UF3 under (a) different light conditions, and (b) different temperatures.

      Changes in antioxidant activities of purified UF3 in in vitro digestion

    • To assess the effect of in vitro enzymatic digestion on the antioxidant activity of purified UF3, the change of hydroxyl radical scavenging activity, DPPH scavenging activity, and reducing power of purified UF3 during the in vitro digestion process were investigated. As presented in Fig. 6a−c, the in vitro enzymatic digestion weakened the antioxidant activity of purified UF3. The antioxidant activities of melanoidins may be directly related to its structure, and in vitro digestion could result in a change in the structure of purified UF3. There are two reasons for this: firstly, the generation of new non-covalently bound melanoidin compounds in the presence of digestive enzymes, and secondly, the free radical scavenging activity of food melanoidins attributed to the presence of phenolic groups in their structure[32,36]. Although the antioxidant activity of digested samples was lower than that of undigested samples, all digested samples still exhibited a good antioxidant activity. After in vitro intestinal digestion, the hydroxyl radical scavenging activity, DPPH scavenging activity, and reducing power of digested UF3 was 65%, 50%, and 0.33, respectively. It could be seen that the purified UF3 extracted from BLB has potential antioxidant application value in vivo.

      Figure 6. 

      The changes on antioxidant activities of purified UF3 in vitro digestion. (a) Hydroxyl radical scavenging activity, (b) DPPH scavenging activity, and (c) reducing power. Different letters indicate significant difference at p < 0.05.

    Conclusion

    • This study demonstrated that melanoidin fractions with LMW (3 kDa < MWC < 10 kDa) were dominant fractions in low thermal induced BLB, and the dominant fractions exhibited excellent antioxidant activities. The dominant melanoidin fractions (UF3) were purified, preliminarily characterized, and its antioxidant activities checked by in vitro digestion. The results of FTIR spectroscopy revealed that purified UF3 showed a typical structure of melanoidins. The optimum purification conditions proved to be efficient for the purification of UF3 from BLB. The purified UF3 was stable in the dark and under light conditions without ultraviolet rays. However, the purified UF3 with LMW was unstable under heat treatment. The antioxidant activities of purified UF3 decreased after in vitro intestinal digestion, yet it exhibited a certain level of antioxidant activity. These results suggest that the UF3 extracted from low thermally induced BLB could be considered as a potential antioxidant ingredient and as a potential antioxidant component for the development of orally consumable functional foods.

    Author contributions

    • The authors confirm contribution to the paper as follows: study conception and design: Chen J; experimental processing: Wang J, Liu Y; data collection and validation: Wang J, Li H; analysis and interpretation of results: Li H, Wang W; draft manuscript preparation: Chen J, Pan Y; language editing, manuscript revision: Hu Y. All authors reviewed the results and approved the final version of the manuscript.

    Data availability

    • Data are available from the corresponding author on reasonable request.

      Acknowledgments

      • This study was partly supported by the Innovative Team of Modern Agricultural Industry Technology System in Tianjin (ITTHRS2021000) and Key R & D Project of Xinjiang Uygur Autonomous Region (2022B02030).

      Conflict of interest

      • The authors declare that they have no conflict of interest.

      • Authors contributed equally: Junran Chen, Jie Wang

      Supplementary information
      • Supplemental File 1 Screening of macroporous adsorption resin for purification of melanoidins from low thermal induced black Lycium barbarum L.
      Rights and permissions
      • Copyright: © 2024 by the author(s). Published by Maximum Academic Press on behalf of China Agricultural University, Zhejiang University and Shenyang Agricultural University. This article is an open access article distributed under Creative Commons Attribution License (CC BY 4.0), visit https://creativecommons.org/licenses/by/4.0/.
    Figure (6)  Table (1) References (36)
  • About this article
    Cite this article
    Chen J, Wang J, Liu Y, Li H, Wang W, et al. 2024. Study on physicochemical properties and antioxidant activities of melanoidins extracted from low thermal induced black Lycium barbarum L. Food Innovation and Advances 3(3): 288−294 doi: 10.48130/fia-0024-0027
    Chen J, Wang J, Liu Y, Li H, Wang W, et al. 2024. Study on physicochemical properties and antioxidant activities of melanoidins extracted from low thermal induced black Lycium barbarum L. Food Innovation and Advances 3(3): 288−294 doi: 10.48130/fia-0024-0027
    shu

Catalog

  • HTML

  • Introduction
  • Materials and methods

  • Materials and preparation of heat-induced black Lycium barbarum

  • Preparation of melanoidins

  • Isolation and evaluation of melanoidin fractions

  • Orthogonal experimental design of resin purification

  • Determination of the chemical constituents of melanoidin fractions

  • Antioxidant activity determination

  • Hydroxyl radical scavenging activity
  • Ferric-reducing antioxidant power
  • DPPH radical scavenging activity

  • Fourier transform infrared spectroscopy (FTIR)

  • Stability assay

  • Antioxidant activity in vitro simulated digestion

  • Statistic analysis

  • Results and discussion

  • Isolation and preliminary evaluation of melanoidin fractions

  • Content of isolated melanoidin fractions
  • Chemical compositions of isolated melanoidin fractions
  • Antioxidant activities of isolated melanoidin fractions

  • Optimization of UF3 purification conditions

  • FTIR analysis of purified UF3

  • Stability of purified UF3

  • Changes in antioxidant activities of purified UF3 in in vitro digestion

  • Conclusion
  • Author contributions
  • Data availability
  • Acknowledgments
  • Conflict of interest
  • Supplementary information
  • Rights and permissions
  • About this article
logo

Contents

Policies

Services

Partnerships

© 2025 Maximum Academic Press, except Open Access articles. MAP is a member of ALPSP, COPE, CrossRef, cOAlition S, OASPA and STM.

Privacy Policy  |  Site Map  |  Terms and Conditions

/

DownLoad:  Full-Size Img  PowerPoint
Return
Return