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Stability of citral in oil-in-water emulsions protected by a soy protein–polysaccharide Maillard reaction product
Food Research International 69 (2015) 357–363
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Stability of citral in oil-in-water emulsions protected by a soy protein–polysaccharide Maillard reaction product Yuexi Yang a,b, Steve Cui a,b, Jianhua Gong b, S. Shea Miller b, Qi Wang b,⁎, Yufei Hua a,⁎⁎ a b
State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi, Jiangsu Province 214122, PR China Guelph Food Research Centre, Agriculture and Agri-Food Canada, Guelph, Ontario N1G 5C9, Canada
a r t i c l e
i n f o
Article history: Received 11 September 2014 Accepted 10 January 2015 Available online 15 January 2015 Keywords: Citral Stability Emulsion Soy protein–polysaccharide Maillard reaction product Simulated gastrointestinal condition
a b s t r a c t Citral is an important essential oil with antibacterial activities, but its use as an antibiotic alternative is limited due to its physical and chemical instability during processing and in biological systems such as the gastrointestinal tract of animals. This study aimed to investigate the capacity of a soy protein–polysaccharide Maillard reaction product (SPPMP) to stabilize citral in an oil-in-water emulsion system. The retention rates of citral in the emulsions during long time storage, upon heating and under simulated gastrointestinal conditions were determined. The results showed that SPPMP-stabilized emulsions demonstrated outstanding ability to stabilize citral under all challenge conditions as compared to emulsions stabilized by soy protein only, or by physical mixtures of soy protein and polysaccharide. Therefore, SPPMP-stabilized emulsions could potentially be used as protectors and carriers for targeted delivery of citral or other hydrophobic compounds to animal/human intestines. © 2015 Published by Elsevier Ltd.
1. Introduction Citral is a major component of lemon essential oils with strong lemon flavor, which is traditionally used as a flavoring agent. Essentially, citral is a mono-terpene aldehyde composed of two geometrical isomers, neral and geranial, at a ratio of approximately 2:3 (Kimura, Doi, Iwata, & Nishimura, 1981). Citral exhibited a broad spectrum of antimicrobial activities against both Gram-positive and Gram-negative bacteria and fungi in in vitro tests (Onawunmi, 1989; Saddiq & Khayyat, 2010; Stevens, Jurd, King, & Mihara, 1971). Recently, citral has been proposed as a potential alternative to antibiotics in animal feed for prevention of bacterial infections. In this context, targeted delivery of citral to the animal intestines is likely to enhance its antibacterial efficacy because the intestine is a major reservoir of many foodborne pathogens. Citral also holds potential to be used as a natural preservative in food formulations. Currently, the use of citral as an antimicrobial agent in the food or feed industry is limited by several factors, including its chemical instability, high hydrophobicity, and volatility. Owing to its unsaturated structure, citral tends to undergo chemical transformation leading to loss of its functionality (Kimura, Iwata, & Nishimura, 1982; Kimura, Nishimura, Iwata, & Mizutani, 1983a; Kimura et al., 1981; Schieberle, Ehrmeier, &
Abbreviations: SPI, soy protein isolate; SSPS, soy soluble polysaccharide; SPP, mixture of soy protein isolate and soy soluble polysaccharide; SPPMP, soy protein–polysaccharide Maillard reaction product; SGF, simulated gastric fluids; SIF, simulated intestinal fluids. ⁎ Corresponding author. Tel.: +1 226 217 8077; fax: +1 226 217 8181. ⁎⁎ Corresponding author. Tel./fax: +86 510 85917812. E-mail addresses: [email protected] (Q. Wang), [email protected] (Y. Hua).
http://dx.doi.org/10.1016/j.foodres.2015.01.006 0963-9969/© 2015 Published by Elsevier Ltd.
Grosch, 1988). For example, in acidic conditions, citral can undergo a series of cyclization, oxidation and dehydration reactions, producing various transformation products including p-mentha-dien-4-ol, p-menthadiene-8-ol, p-cymene-8-ol, -p-dimethylstyrene, p-cymeme, p-cresol and p-methylacetophenone, etc (Clark & Chamblee, 1992; Kimura, Nishimura, Iwata, & Mizutani, 1983b; Kimura et al., 1982; Peacock & Kuneman, 1985; Schieberle & Grosch, 1988; Ueno, Masuda, & Ho, 2004; Wolken, ten Have, & van der Werf, 2000). At neutral and alkaline conditions, citral could undergo amino acid-catalyzed conversion to produce 6-methyl-5-hepten-2-one and acetaldehyde (Wolken et al., 2000). Animal studies from our group have shown that only a small fraction of orally administered citral could reach the lower intestine of chickens (data to be published separately). The loss of citral would be partially owing to the transformation discussed above, as well as absorption in the upper gastrointestinal tract. Previous studies suggest that the chemical stability of citral could be improved by emulsification. At both neutral and acidic conditions, citral exhibited a greater resistance to chemical transformation when presenting in emulsion droplets than presenting in aqueous phase (Choi, Decker, Henson, Popplewell, & McClements, 2009, 2010a, 2010b; Djordjevic, Cercaci, Alamed, McClements, & Decker, 2007, 2008; Mei et al., 2010; Yang, Tian, Ho, & Huang, 2011b; Zhao, Ho, & Huang, 2013). However, no report has been found in the literature to date regarding the performance of emulsified citral under gastrointestinal conditions. Recently, protein–polysaccharide Maillard reaction products were found to be effective in stabilizing emulsion systems, especially upon heating and under acidic condition (Akhtar & Dickinson, 2003;
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Dickinson & Semenova, 1992; Kasran, Cui, & Goff, 2013). In our previous study, a Maillard reaction product (SPPMP) was prepared from soy protein isolates and soy polysaccharides which exhibited superior emulsification properties in stabilizing a citral-loaded emulsion in a prolonged storage period, upon heating and during simulated gastrointestinal digestion (Yang et al., 2014). However, the chemical stability of citral in such an emulsion system under these conditions remains to be evaluated before it can be considered as a potential carrier for intestinal delivery of citral, and other applications in food formulation. Therefore, the objective of the current study was to evaluate the stability of citral in a SPPMP-stabilized emulsion system under challenge conditions, including prolonged storage, heating and simulated gastrointestinal digestion. 2. Materials and methods 2.1. Materials Soy protein isolate (SPI) and soy soluble polysaccharide (SSPS) were provided by Shandong Gushen Industrial and Commercial Co., Ltd (Shandong, China). The sample of SPI contained 92% protein, whereas the sample of SSPS contained 85% polysaccharides with an average molecular weight of 54.2 kDa. Citral (mixture of cis and trans isomers, 95% pure), pepsin (from porcine gastric mucosa powder, ≥ 250 units/mg solid), pancreatin (from porcine pancreas, composed of amylase, trypsin, lipase, ribonuclease and protease), and bile extract (porcine) were purchased from Sigma Aldrich Chemical Co. (St. Louis, MO, USA). Hexane (HPLC grade) was purchased from Caledon Laboratories Ltd (Georgetown, ON, Canada). All other reagents and chemicals were of analytical grade. 2 M HCl or 2 M NaOH solutions were used to adjust pH. Ultrapure water produced by a Milli-Q® Reference Water Purification System (Lit No: PB0015ENUS) was used to prepare all solutions. 2.2. Preparation of SPP and SPPMP A procedure and an optimized formulation developed from our previous study were used to prepare the soy protein–soy polysaccharide mixture (SPP) and their Maillard reaction product (SPPMP) (Yang et al., 2014). Briefly, the SSPS and SPI solutions were mixed at SSPS:SPI ratio of 3:5 (dry weight) and freeze dried to yield soy protein–polysaccharide mixture (SPP). To prepare SPPMP, a portion of SPP was incubated at 60 °C and relative humidity of 75% for 72 h, and then dissolved into water with stirring for 4 h, followed by centrifugation at 15,000 g for 30 min. The supernatant was collected and freeze dried, yielding the soy protein–soy polysaccharide Maillard reaction product (SPPMP). Above prepared SPP and SPPMP were sealed in bottles and stored at 4 °C until further use. 2.3. Preparation of citral-loaded emulsions stabilized by SPI, SPP and SPPMP The citral-loaded emulsions stabilized by SPI, SPP and SPPMP were prepared according to Yang et al. (2014). SPPMP-stabilized emulsions contained 10% (w/w) citral and 2.0%, 2.5%, 3.3% and 5.0% of SPPMP, respectively, which corresponded to the ratio of citral to SPPMP (w/w) at 5:1, 4:1, 3:1 and 2:1. Portions of the emulsions were adjusted to pH 7.0 or 3.0 with 2 M HCl with stirring at 250 rpm for 30 min. The citralloaded emulsions stabilized by SPP or SPI contained 10% citral (w/w) with the ratio of citral to SPP or SPI at 3:1 only. Triplicates were prepared for each emulsion. 2.4. Storage and heat treatment of emulsions Freshly made emulsions were stored in 20 mL capped amber glass vial with approximately 10 mL headspace in the dark at 25 °C. Some of the freshly made emulsions were heat-treated at 90 °C for 30 min
before storage, according to Xu and Yao (2009) to mimic a typical sterilization treatment in food processing.
2.5. Exposure to simulated gastrointestinal conditions Simulated gastric fluids (SGF) were prepared at pH 1.5, 2.0 and 2.5 containing 0.32% (w/w) pepsin and 0.2% (w/w) NaCl (Sarkar, Goh, Singh, & Singh, 2009). Simulated intestinal fluids (2×) were prepared at pH 7.5 containing 20 g/L pancreatin, 5 g/L bile salts, 80 mM K2HPO4, 150 mM NaCl and 40 mM CaCl2 according to Sarkar et al., with slight modifications (Sarkar, Horne, & Singh, 2010). Each freshly made emulsion was mixed with SGF (1:20, v/v) at 37 °C and divided into several aliquots stored in 10 mL amber glass tubes with continuous shaking at 100 rpm for 2 h. At each time point of 0, 0.5, 1.0, 1.5 and 2 h, three tubes from each treatment were removed from the incubation bath and the peptic digestion was stopped by adjusting to pH 7.5 with 2 M NaOH (Fafaungwithayakul et al., 2011), then cooled to 25 °C for GC analysis (after hexane extraction as described in Section 2.6). For the remaining tubes containing the emulsions after exposure to SGF, 2 M NaOH was added to adjust the pH to 7.5. Two models were adopted in the incubation under simulated intestinal conditions following the SGF incubation. In the first model, an equal volume of SIF (2 ×) was added to the emulsions after SGF incubations at pH 7.5, then incubated at 37 °C with shaking at ~ 100 rpm for 4 h. In the second model, the digesta from 12-day-old broilers (ileal digesta and jejunal digesta at a weight ratio of 1:1) was used instead of SIF at the same volume ratio, with the pH adjusted to 7.5. After incubation in simulated intestinal conditions for 4 h, samples were removed from the incubation bath, and cooled to 25 °C for GC analysis as described above.
2.6. Gas chromatography (GC) and gas chromatography–mass spectrometry (GC-MS) To measure the concentration of citral in an emulsion, approximately 2 mL of emulsion sample was mixed with hexane at 4 times the volume, and vortexing at speed of 1000 rpm for 30 min, then allowed to stand for another 30 min. The supernatant in each tube was collected and filtered through a syringe-driven filter unit (polytetrafluoroethylene, 0.22 μm), diluted to a citral concentration range of 20–100 μg/mL with hexane, and then analyzed by GC. An Agilent 6890 Series GC System equipped with a flame ionizing detector (FID) and a fused silica capillary column (30 m × 0.25 mm × 0.25 μm film thickness; temperature limits: 35–280 °C; SUPELCO WAX ™ 10) was used. The oven temperature was programmed as follows: initial temperature 60 °C (held for 1 min) followed by an increase at 8 °C/min to 240 °C and held for 1 min; injector and detector temperatures were 250 °C. Helium (2.0 mL/min) was used as the carrier gas. A sample (1.0 μL) was injected into the GC for testing and the two isomers of citral were identified by comparison to retention times with the analytical standard of citral from Sigma. The quantification of citral was conducted by determining the peak areas of neral and geranial, respectively, in the GC-FID graph and calculating against a standard curve made from the citral standard. Citral concentration was expressed as the sum of the two isomers. The degradation products of citral were identified by GC-MS. Electron impact (EI) mass spectrometric data were collected using a Hewlett–Packard 5973 mass spectrometer interfaced to a Hewlett– Packard 6890 gas chromatograph. The operating conditions of the mass spectrometer were as follows: split ratio 1:50, ionization voltage 70 eV and ion source temperature 140 °C. The operating conditions of the gas chromatograph were the same as in the GC-FID method. The mass spectrum of each identified compound was compared to the actual retention time and mass spectrum of each compound stored in the relevant standard databases (Wiley Registry® of Mass Spectral Data and NIST Mass Spectrometry Data Centre).
Y. Yang et al. / Food Research International 69 (2015) 357–363
100 Retention rate of citral (%)
2.7. Evaluation of citral stability The stability of citral in oil-in-water emulsions was evaluated by measuring the concentration of each of its isomers under different experimental conditions, and calculated as retention rate relative to day 0. Total retention rate of citral was calculated as the sum of the concentration of the two isomers at different experimental conditions relative to day 0. The day 0 concentrations were recorded as the concentration of citral isomers initially added to the emulsions. 2.8. Statistical analysis
3. Results and discussion 3.1. Chemical stability of citral in emulsions as influenced by citral to SPPMP ratio The chemical stability of citral was found to be dependent on the stability of SPPMP-stabilized emulsions, both of which varied with the citral to SPPMP ratio in the formulation. As shown in Table 1, increase of citral to SPPMP ratio resulted in an increase of average droplet size (Z-average) and polydispersity index (PDI) in the emulsions at the same pH. Increase of droplet size and polydispersity often associates with decrease in emulsion stability (Ray & Rousseau, 2013). At citral to SPPMP ratio of 4:1 and above, free citral was visible on the surface of the emulsion, indicating insufficient SPPMP to fully cover the citral droplets. As shown in Fig. 1, the retention rate of citral in SPPMPstabilized emulsions after 14-day-storage at neutral pH and 25 °C increased from 85.1% to 92.2% when the proportion of SPPMP was increased (from 5:1 citral:SPPMP to 2:1 citral:SPPMP). A similar trend was observed in the SPPMP-stabilized emulsions at pH 3.0 under the same storage conditions. Choi et al. (2010b) also reported that the retention rate of citral increased (from 25% to 54%) as the concentration of emulsifier (Tween 80) increased (from 0% to 5%) after one week storage at 20 °C and pH 3.0. The stability of citral was poorer, and retention rates were lower when emulsions were stored at pH 3.0 compared to pH 7.0 at all citral to SPPMP ratios. This observation is in agreement with the previous studies, in which the retention rate of citral was reduced as the pH decreased from 7.0 to 3.0 (Choi et al., 2009, 2010a; Mei et al., 2010). The difference between the retention rates of citral in SPPMP-stabilized emulsions at pH 3.0 and pH 7.0 became more apparent as the ratio of citral to SPPMP increased. Previous studies have demonstrated that citral inside emulsion droplets exhibited better chemical stability compared to free citral in the aqueous phase (Choi et al., 2009; Djordjevic et al., 2007, 2008); therefore, the chemical stability of citral in citral-loaded oil-in-water emulsions could be enhanced Table 1 The mean particle diameter and polydispersity index of citral-loaded emulsions with various ratios of citral to SPPMP after storage at 25 °C for 14 days.
7
3
a,b,c,d,e,f
Ratio
Mean particle diameter
(citral:SPPMP)
(Z-average, nm)
5:1 4:1 3:1 2:1 5:1 4:1 3:1 2:1
938 ± 44b 791 ± 25c 534 ± 14de 487 ± 9f 1064 ± 42a 825 ± 29c 561 ± 16d 528 ± 11e
Polydispersity index
0.47 ± 0.04a 0.32 ± 0.02b 0.21 ± 0.01d 0.18 ± 0.004e 0.45 ± 0.07a 0.36 ± 0.03b 0.24 ± 0.01c 0.21 ± 0.007d
Different letters in each column indicate significant difference (p ≤ 0.05).
90
pH=7.0
pH=3.0 c
bc
b
b
b
a
a
80
d
70 60 50 5:1
4:1
3:1
2:1
Ratio (citral: SPPMP) Fig. 1. Effects of citral to SPPMP ratio on the total retention rate of citral in SPPMP-stabilized emulsions after storage at pH 7.0 and 3.0 for 14 days at 25 °C. Different letters indicate significant difference (p ≤ 0.05).
with the improvement of emulsion stability. In a previous study, gum arabic (GA)-stabilized emulsions (5% GA, 10% citral, w/w) exhibited outstanding capacity to stabilize citral during storage, better than whey protein isolate (WPI)-stabilized emulsions (5% WPI, 10% citral, w/w) (Djordjevic et al., 2008). Compared with GA, SPPMP showed superior capacity for citral stabilization, especially at low pH. For example, after storage at 20 °C for 14 days, the retention rate of citral was 80% (pH 3.0) and 85% (pH 7.0) in the GA-stabilized emulsions (Djordjevic et al., 2008). Whereas, after storage for 14 days at a higher temperature (25 °C), the retention rate of citral was 92% (pH 3.0) and 90% (pH 7.0) in SPPMP-stabilized emulsions. As there was no significant difference (p N 0.05) between the stability of citral in emulsions with citral to SPPMP ratio at 3:1 and 2:1, the ratio of 3:1 was chosen for the rest of this study due to the higher citral content in the emulsion. 3.2. Citral stability in emulsions as influenced by storage time and heating The stability of citral in the SPI, SPP and SPPMP stabilized emulsions was very different (p ≤ 0.05), as indicated by the total retention rates of citral at each time point shown in Fig. 2. The SPPMP-stabilized emulsions showed superior ability to inhibit the loss of citral, yielding the highest retention rates at all tested time points in the storage period. The total retention rate of neral and geranial in GA-stabilized emulsions was about 80% when stored at pH 7.0 and 20 °C for 35 days, reported by Djordjevic et al. (2008), which is similar to our observed total retention
SPl
SPP
SPPMP
SPI (heating)
SPP (heating)
SPPMP (heating)
100 Retention rate of citral (%)
The statistical analysis was performed using Design-expert (Designexpert Version 9, Stat-Ease Int) software. Analysis of variance (ANOVA) was performed on the data, and a least significant difference (LSD) test with a confidence interval of 95% was used to compare the means.
pH of emulsion
359
75
50
25 0
30
60
90
120
150
Time (days) Fig. 2. Retention rate of citral in heated and unheated emulsions stabilized by SPI, SPP and SPPMP at pH 7.0 during storage at 25 °C.
Y. Yang et al. / Food Research International 69 (2015) 357–363
rate of neral and geranial in SPP-stabilized emulsion under similar storage conditions. When the SPPMP-stabilized emulsions were stored at higher temperature (25 °C), the total retention rate of neral and geranial was 85% after storage for 35 days and around 80% after storage for 70 days. In addition, SPPMP reduced the negative impact of heating on citral stability. In SPPMP-stabilized emulsions, a loss of 2.4 ± 0.8% of citral was found immediately after heating. In contrast, the loss rates of citral were found to be 5.0 ± 0.9% and 7.1 ± 1.6% for SPP- and SPIstabilized emulsions, respectively. It was also observed that the difference in retention rate between heated and unheated emulsions was significantly smaller for SPPMP-stabilized emulsions as compared to SPI- and SPP-stabilized emulsions in a prolonged storage period (p ≤ 0.05). After storage at 25 °C for 140 days, there were still 63.6 ± 2.3% and 69.3 ± 2.0% of citral remaining in the heated and unheated SPPMP-stabilized emulsions, respectively. The high stability of citral in the SPPMP emulsion could be attributed partially to the high stability of SPPMP-stabilized emulsions. It is widely acknowledged that the breakdown of emulsion and release of core material are usually associated with larger droplet size (Ray & Rousseau, 2013). The average particle size in SPPMP-stabilized emulsions was smaller than those in SPI-/SPP-stabilized emulsions during storage up to 140 days (p ≤ 0.05). In addition, the droplet sizes in the SPPMPstabilized emulsions did not change much after heat treatment and during the subsequent 2 months of storage at room temperature (Yang et al., 2014). The improved stability of citral by incorporation of SSPS into SPI-based emulsions could also be partially attributed to the reduction of negative surface charge and protein/amino acids content in the emulsions; the lowest negative surface charge and protein content were observed in the SPPMP-emulsions at neutral pH as compared to SPI- and SPP-emulsions (Yang et al., 2014). At neutral pH, ζ-potential of citral-loaded emulsion droplets stabilized by SPPMP was − 31.1 ± 0.5 mV, while those stabilized by SPP and SPI was − 37.6 ± 0.4 mV and − 46.2 ± 0.3 mV, respectively. The reduction of negative surface charge and protein/amino acid content in citral-loaded emulsions could slow the de-stabilization of citral. As mentioned earlier, polymers with high negative charges potentially attract more cations, such as iron ions, which could promote the oxidative transformation of citral (Choi et al., 2010a; Djordjevic et al., 2007; Hu, McClements, & Decker, 2003; Mei et al., 2010); whereas the presence of protein/amino acids at neutral pH could catalyze the de-acetylation of citral (Wolken et al., 2000). Several previous studies have also evaluated the capabilities of various emulsion systems to inhibit the transformation of citral, but few of them focused on long-term storage and the effect of heating, which are important factors to be considered for the applications of such systems. The results in the current study showed that the retention rate of citral was higher in SPPMP-stabilized emulsions during storage, compared to commonly used emulsifiers including sodium dodecyl sulfate, lauryl alginate, gum arabic, whey protein isolate, and soy protein isolate (Choi et al., 2010a; Djordjevic et al., 2007, 2008).
3.3. Exposure to simulated gastric and intestinal conditions 3.3.1. The stability of citral in simulated gastric conditions Maintaining the stability of citral at low pH is a major challenge for some applications, such as in intestinal delivery of citral as an antimicrobial agent and in acidic beverages as a flavoring and preservative agent (Maswal & Dar, 2014; Zhao et al., 2013). Although a number of investigations have been carried out on the stability of citral in acidic emulsions with various emulsifiers and/or antioxidants, none of them tested the stability of citral under gastric and intestinal conditions. The stomach is a major bioreactor for digestion of food, with strong acidity, proteolytic activity and vigorous agitation (Kong & Singh, 2010; Sarkar et al., 2009; Singh, Ye, & Horne, 2009). In this study, citral-loaded emulsions were exposed to SGF (0.32% pepsin, 0.2% NaCl, w/w, pH 2.0) with slow stirring at 37 °C, which mimics the environment of the stomach.
Because the retention rates of neral and geranial changed differently with time during incubation in SGF, they are expressed separately in Fig. 3. Of the three types of emulsions tested, the stability of citral in SGF was highest in SPPMP-emulsions. Previous studies have demonstrated that in an oil-in-water emulsion system, citral located outside the droplets was more susceptible to transformation, compared with that located inside the droplets (Choi et al., 2009, 2010a; Mei et al., 2010). In other words, the stability of citral in an oil-in-water emulsion was inversely associated with the release of citral from emulsion droplets: higher release rate of citral to the aqueous phase leads to lower stability of citral. The observations from the current study and our previous study (Yang et al., 2014) are in agreement with this conclusion. In our previous study, the release rate of citral was found to be the lowest in SPPMP-emulsions compared with SPI- and SPP-emulsions. In general, neral was less stable than geranial in SGF. However, the difference in retention rates between neral and geranial was not significant in SPPMP-emulsion after 2 h incubation in SGF. In contrast, the differences were found to be significant for SPI- and SPP-emulsions after 30 and 60 min incubation in SGF, respectively (p ≤ 0.05); the observed difference increased with incubation time. This result agrees well with an earlier report that the transformation of neral was faster than geranial (p ≤ 0.05) in gum arabic-stabilized emulsions during storage under acidic conditions (pH 3.0) (Djordjevic et al., 2008). In acyclic systems, trans isomers are usually more stable than cis isomers, which is due to increased unfavorable steric interactions in the cis isomer (March, 1992). Fig. 4a shows a GC chromatogram of citral before emulsification; Fig. 4b shows a GC chromatogram of citral and its transformation products in emulsions stabilized by SPI, SPP and SPPMP after SGF incubation. In comparison with Fig. 4a, two new peaks appeared in Fig. 4b at retention times of 11.5 min and 12.4 min, respectively. These were identified by GC-MS analysis as p-mentha-dien-4-ol and p-mentha-diene-8ol, the major products from the first stage of acid-catalyzed transformation of citral (Kimura et al., 1982; Peacock & Kuneman, 1985). Much smaller peaks at retention times of 13.4 min and 14.6 min (data not shown) were identified as the further oxidation products of pmentha-dien-4-ol and p-mentha-diene-8-ol: -p-dimethylstyrene and p-methylacetophenone (second-stage transformation products of citral) respectively (Kimura et al., 1983b; Peacock & Kuneman, 1985; Schieberle & Grosch, 1988). These oxidation products were also previously identified as the main transformation products of citral in citralcontaining solutions or citral-loaded emulsions stored at 25–40 °C for 7–40 days at pH 3.0. (Liang, Wang, Simon, & Ho, 2004; Maswal & Dar, 2013; Ueno et al., 2004; Yang et al., 2011b; Zhao et al., 2013). It was
SPPMP geranial SPPMP neral
SPP geranial SPP neral
SPI geranial SPI neral
100 Retention rate of citral (%)
360
90
* * *
80
* * *
70
*
60 0
30
60
90
120
Time (min) Fig. 3. Retention rate of neral and geranial in emulsions stabilized by SPI, SPP and SPPMP during incubation in SGF at pH 2.0. *indicates significant difference between geranial and neral in the same emulsions at the same time point (p ≤ 0.05).
Y. Yang et al. / Food Research International 69 (2015) 357–363
a
361
100 120
Neral
Retention rate of citral˄%˅
FID response (PA)
100 80 60 40 20 0 10.5
11
11.5
12
12.5
13
Neral (heating)
Geranial
e
90
de
d
a
cd
cd
c
80
Geranial (heating)
de cd
ac a
b
70
13.5
Retention time (min)
60 1.5
b
2.0
2.5
pH 100 SPPMP SPP SPI
FID response (PA)
80
Fig. 5. Retention rates of neral and geranial in heated and unheated SPPMP-stabilized emulsions after incubation in SGF at different pH for 2 h. Different letters indicate significant difference (p ≤ 0.05).
60
40
20
0 10.5
11
11.5
12
12.5
13
13.5
Retention time (min)
Fig. 4. Gas chromatogram: (a) citral before emulsification; (b) citral and its transformation products in emulsions stabilized by SPI, SPP and SPPMP after SGF incubation at pH 2.0 for 2 h.
reported that while the retention rate of citral decreased from 80% to 0%, the concentration of -p-dimethylstyrene and p-methylacetophenone (second-stage transformation products of citral) increased by 6-fold (Liang et al., 2004). In contrast to the previous studies, the transformations of citral after SGF incubation at pH 2.0 mostly remained at the first stage in the current study, due to the short incubation time (2 h) and the protection provided by the emulsion. The concentrations of pmentha-dien-4-ol, p-mentha-diene-8-ol and their oxidation products were lowest in the SPPMP-stabilized emulsions, compared with SPIand SPP-stabilized emulsions (Fig. 4b). This result suggests that SPPMP-stabilized emulsions could effectively retard the transformation of citral in simulated gastric conditions. 3.3.2. The influence of pH of SGF and pre-heating of emulsions For live animals, the pH of the stomach fluids varies from pH 1 to 3 depending on the digestive status (i.e. fullness, appetite). In this study, SPPMP-stabilized emulsions (heated and unheated) were tested in SGF at pH 1.5, 2.0 and 2.5. The stability of the citral in unheated SPPMP-stabilized emulsions was found to be responsive to the pH of SGF (Fig. 5). The retention rate (p ≤ 0.05) of geranial decreased significantly as the pH values decreased, whereas for neral, the retention rate did not significantly change as the pH decreased from 2.5 to 2.0. The retention rate between neral and geranial showed significant differences only as the pH decreased to 1.5 (p ≤ 0.05). The higher concentration of H+ found at lower pH in an emulsion system would be the main factor that is responsible for the transformation of citral because H+ participates in the acid-induced transformation of citral to form monoterpene alcohols (Kimura et al., 1982). This was confirmed by more rapid transformation of citral in an emulsion system at pH 3.0, compared with that at pH 7.0 by Yang, Tian, Ho, and Huang (2011a). Although the retention rate of citral also decreased in SPPMP-stabilized
emulsions as pH decreased, the extent was limited. The retention rate of citral in SPPMP-stabilized emulsions after SGF incubation at pH 1.5 was calculated to be 10% higher than the retention rate of citral in SPP-stabilized emulsions at the same pH (data not shown), and 4% higher than the retention rate of citral in SPP-stabilized emulsions at pH 2.0 (data from Figs. 5 and 3). Fig. 5 also shows the influence of heat treatment on the stability of citral in SPPMP-stabilized emulsions during SGF incubation. The difference in the retention rate of citral between heated and unheated emulsions during SGF incubation was somewhat dependent on the pH of SGF; the difference in retention rates of citral (neral and geranial) between heated and unheated SPPMP-stabilized emulsions was only significant at pH 1.50 (p ≤ 0.05). In contrast, for SPI- or SPP-stabilized emulsions, the retention rates of citral in the heated emulsions were significantly decreased, compared to the unheated emulsions at pH 1.5, 2.0 and 2.5 during SGF incubation (data not shown). The stability of citral in an emulsion system during SGF incubation would be related to the pepsin-digestibility of the emulsifiers. The pepsin-digestibility of SPI was shown to increase after heat treatment (Tsumura, Saito, Kugimiya, & Inouye, 2004). In heat-treated emulsions, the increase of pepsin-digestibility of SPI could result in disintegration of emulsion droplets and the release of citral during SGF incubation. On the contrary, pepsin-digestibility of SPPMP might be decreased, as the SPPMPstabilized emulsion exhibited enhanced stability under gastric conditions and better resistance to heat treatment (Yang et al., 2014). Therefore, the improved chemical stability of citral in a SPPMP-stabilized emulsion can be attributed mainly to the enhanced stability of the emulsion system. 3.3.3. Exposure to simulated gastro-intestinal tract Two in vitro models were chosen for the current study. In the first model, the emulsions were exposed to simulated intestinal fluid (SIF) after 2 h SGF incubation; in the second model, the intestinal digesta collected from 12-day-old chicks was used instead of SIF. The total retention rates of citral in SPPMP-stabilized and SPPstabilized emulsions were about 1.5 and 1.3 times higher, respectively, than that in SPI-stabilized emulsions after incubation in simulated gastrointestinal fluids (Fig. 6). Consequently, the concentration of citral transformation products was highest in SPI-stabilized emulsions and lowest in SPPMP-stabilized emulsions (Fig. 7). Compared with the corresponding emulsion after incubation in SGF only (Fig. 4b), the concentration of citral transformation products increased after SGF-SIF incubation (Fig. 7). A similar trend was observed for the emulsions incubated with chicken digesta as shown in Fig. 6, although the
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90 Retention rate of citral (%)
SIF
4. Conclusions
Digesta
80
Incorporation of SSPS could enhance the stability of citral in SPIbased emulsion systems. Compared with soy protein–polysaccharides mixture (SPP), soy protein–polysaccharide Maillard reaction products (SPPMP) offered significantly better protection to citral in a prolonged storage period, upon heat treatment, and in simulated gastrointestinal tracts. The high emulsification ability and stability of SPPMP helped to protect citral from interaction with compounds in the aqueous phase and other environmental factors that could induce the transformation of citral and cause loss of its functionality. The results from the present study suggest that the SPPMP-stabilized emulsion system could be useful for the protection and targeted delivery of citral to the small intestine.
c
70
b b
60 a a
50
d
40 30 SPI
SPP
SPPMP
Fig. 6. Retention rate of citral in emulsions stabilized by SPI, SPP and SPPMP after incubation in simulated gastro-intestinal conditions with SIF or digesta. Different letters indicate significant difference (p ≤ 0.05).
corresponding retention rates were significantly lower compared to those in SIF. Further loss of citral in SIF and the digesta is mainly due to the breakdown of emulsions and subsequent digestion (Yang et al., 2014). Soy proteins are subject to hydrolysis by proteases present in the SIF and the digesta, which may lead to disintegration of emulsion droplets. Incorporation of SSPS to SPI, either by physical interactions or covalent-links, may spatially shield peptide bonds against proteolysis, thus retard the release of citral from the droplets. Our previous study (Yang et al., 2014) confirmed that the release rate of citral from SPPMP-stabilized emulsions was significantly slower during exposure to SIF, than that from SPI- and SPP-stabilized emulsions. The current results indicate that the SPPMP-stabilized emulsion was the most effective in preventing the loss of citral under simulated gastrointestinal conditions among the three emulsions tested. It is not surprising that the retention rate of citral was lower in chicken digesta than that in SIF. It is well known that there are many kinds of chemical compounds in the digesta. Among those, metal cations, amino acids and other oxidants could interact with the released citral, inducing its transformation. Other macromolecules and small molecules could influence the stability of citral by different mechanisms. In addition, the concentration and locations of citral transformation products may also impact the rate and extent of further transformation of citral in emulsion systems (Choi et al., 2010a). More studies are needed to investigate the effects of different types of components present in the gastrointestinal tract on the stability of citral and citral-loaded emulsions.
100 SPPMP SPP SPI
FID response (PA)
80
60
40
20
0 10.5
11
11.5
12
12.5
13
13.5
Retention time (min)
Fig. 7. Gas chromatograms of citral and its transformation products in emulsions stabilized by SPI, SPP and SPPMP after SGF incubation at pH 2.0 for 2 h followed by SIF incubation at pH 7.5 for 4 h.
Acknowledgments This research is supported by Agriculture and Agri-Food Canada and Canadian Poultry Research Council under the Growing Forward Program (RBPI# 1896 and #13-1039). We also gratefully acknowledge the financial support received from Doctor Candidate Foundation of Jiangnan University (JUDCF10061). Yuexi Yang is a visiting Ph. D. student funded by China Scholarship Council through the AAFC-MOE Program. We wish to thank Ms. Linda Lissemore of the University of Guelph for conducting the GC-MS analysis and Ms. Marta Hernandez of AAFC for helping with GC analysis.
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Food Research International journal homepage: www.elsevier.com/locate/foodres
Stability of citral in oil-in-water emulsions protected by a soy protein–polysaccharide Maillard reaction product Yuexi Yang a,b, Steve Cui a,b, Jianhua Gong b, S. Shea Miller b, Qi Wang b,⁎, Yufei Hua a,⁎⁎ a b
State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi, Jiangsu Province 214122, PR China Guelph Food Research Centre, Agriculture and Agri-Food Canada, Guelph, Ontario N1G 5C9, Canada
a r t i c l e
i n f o
Article history: Received 11 September 2014 Accepted 10 January 2015 Available online 15 January 2015 Keywords: Citral Stability Emulsion Soy protein–polysaccharide Maillard reaction product Simulated gastrointestinal condition
a b s t r a c t Citral is an important essential oil with antibacterial activities, but its use as an antibiotic alternative is limited due to its physical and chemical instability during processing and in biological systems such as the gastrointestinal tract of animals. This study aimed to investigate the capacity of a soy protein–polysaccharide Maillard reaction product (SPPMP) to stabilize citral in an oil-in-water emulsion system. The retention rates of citral in the emulsions during long time storage, upon heating and under simulated gastrointestinal conditions were determined. The results showed that SPPMP-stabilized emulsions demonstrated outstanding ability to stabilize citral under all challenge conditions as compared to emulsions stabilized by soy protein only, or by physical mixtures of soy protein and polysaccharide. Therefore, SPPMP-stabilized emulsions could potentially be used as protectors and carriers for targeted delivery of citral or other hydrophobic compounds to animal/human intestines. © 2015 Published by Elsevier Ltd.
1. Introduction Citral is a major component of lemon essential oils with strong lemon flavor, which is traditionally used as a flavoring agent. Essentially, citral is a mono-terpene aldehyde composed of two geometrical isomers, neral and geranial, at a ratio of approximately 2:3 (Kimura, Doi, Iwata, & Nishimura, 1981). Citral exhibited a broad spectrum of antimicrobial activities against both Gram-positive and Gram-negative bacteria and fungi in in vitro tests (Onawunmi, 1989; Saddiq & Khayyat, 2010; Stevens, Jurd, King, & Mihara, 1971). Recently, citral has been proposed as a potential alternative to antibiotics in animal feed for prevention of bacterial infections. In this context, targeted delivery of citral to the animal intestines is likely to enhance its antibacterial efficacy because the intestine is a major reservoir of many foodborne pathogens. Citral also holds potential to be used as a natural preservative in food formulations. Currently, the use of citral as an antimicrobial agent in the food or feed industry is limited by several factors, including its chemical instability, high hydrophobicity, and volatility. Owing to its unsaturated structure, citral tends to undergo chemical transformation leading to loss of its functionality (Kimura, Iwata, & Nishimura, 1982; Kimura, Nishimura, Iwata, & Mizutani, 1983a; Kimura et al., 1981; Schieberle, Ehrmeier, &
Abbreviations: SPI, soy protein isolate; SSPS, soy soluble polysaccharide; SPP, mixture of soy protein isolate and soy soluble polysaccharide; SPPMP, soy protein–polysaccharide Maillard reaction product; SGF, simulated gastric fluids; SIF, simulated intestinal fluids. ⁎ Corresponding author. Tel.: +1 226 217 8077; fax: +1 226 217 8181. ⁎⁎ Corresponding author. Tel./fax: +86 510 85917812. E-mail addresses: [email protected] (Q. Wang), [email protected] (Y. Hua).
http://dx.doi.org/10.1016/j.foodres.2015.01.006 0963-9969/© 2015 Published by Elsevier Ltd.
Grosch, 1988). For example, in acidic conditions, citral can undergo a series of cyclization, oxidation and dehydration reactions, producing various transformation products including p-mentha-dien-4-ol, p-menthadiene-8-ol, p-cymene-8-ol, -p-dimethylstyrene, p-cymeme, p-cresol and p-methylacetophenone, etc (Clark & Chamblee, 1992; Kimura, Nishimura, Iwata, & Mizutani, 1983b; Kimura et al., 1982; Peacock & Kuneman, 1985; Schieberle & Grosch, 1988; Ueno, Masuda, & Ho, 2004; Wolken, ten Have, & van der Werf, 2000). At neutral and alkaline conditions, citral could undergo amino acid-catalyzed conversion to produce 6-methyl-5-hepten-2-one and acetaldehyde (Wolken et al., 2000). Animal studies from our group have shown that only a small fraction of orally administered citral could reach the lower intestine of chickens (data to be published separately). The loss of citral would be partially owing to the transformation discussed above, as well as absorption in the upper gastrointestinal tract. Previous studies suggest that the chemical stability of citral could be improved by emulsification. At both neutral and acidic conditions, citral exhibited a greater resistance to chemical transformation when presenting in emulsion droplets than presenting in aqueous phase (Choi, Decker, Henson, Popplewell, & McClements, 2009, 2010a, 2010b; Djordjevic, Cercaci, Alamed, McClements, & Decker, 2007, 2008; Mei et al., 2010; Yang, Tian, Ho, & Huang, 2011b; Zhao, Ho, & Huang, 2013). However, no report has been found in the literature to date regarding the performance of emulsified citral under gastrointestinal conditions. Recently, protein–polysaccharide Maillard reaction products were found to be effective in stabilizing emulsion systems, especially upon heating and under acidic condition (Akhtar & Dickinson, 2003;
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Dickinson & Semenova, 1992; Kasran, Cui, & Goff, 2013). In our previous study, a Maillard reaction product (SPPMP) was prepared from soy protein isolates and soy polysaccharides which exhibited superior emulsification properties in stabilizing a citral-loaded emulsion in a prolonged storage period, upon heating and during simulated gastrointestinal digestion (Yang et al., 2014). However, the chemical stability of citral in such an emulsion system under these conditions remains to be evaluated before it can be considered as a potential carrier for intestinal delivery of citral, and other applications in food formulation. Therefore, the objective of the current study was to evaluate the stability of citral in a SPPMP-stabilized emulsion system under challenge conditions, including prolonged storage, heating and simulated gastrointestinal digestion. 2. Materials and methods 2.1. Materials Soy protein isolate (SPI) and soy soluble polysaccharide (SSPS) were provided by Shandong Gushen Industrial and Commercial Co., Ltd (Shandong, China). The sample of SPI contained 92% protein, whereas the sample of SSPS contained 85% polysaccharides with an average molecular weight of 54.2 kDa. Citral (mixture of cis and trans isomers, 95% pure), pepsin (from porcine gastric mucosa powder, ≥ 250 units/mg solid), pancreatin (from porcine pancreas, composed of amylase, trypsin, lipase, ribonuclease and protease), and bile extract (porcine) were purchased from Sigma Aldrich Chemical Co. (St. Louis, MO, USA). Hexane (HPLC grade) was purchased from Caledon Laboratories Ltd (Georgetown, ON, Canada). All other reagents and chemicals were of analytical grade. 2 M HCl or 2 M NaOH solutions were used to adjust pH. Ultrapure water produced by a Milli-Q® Reference Water Purification System (Lit No: PB0015ENUS) was used to prepare all solutions. 2.2. Preparation of SPP and SPPMP A procedure and an optimized formulation developed from our previous study were used to prepare the soy protein–soy polysaccharide mixture (SPP) and their Maillard reaction product (SPPMP) (Yang et al., 2014). Briefly, the SSPS and SPI solutions were mixed at SSPS:SPI ratio of 3:5 (dry weight) and freeze dried to yield soy protein–polysaccharide mixture (SPP). To prepare SPPMP, a portion of SPP was incubated at 60 °C and relative humidity of 75% for 72 h, and then dissolved into water with stirring for 4 h, followed by centrifugation at 15,000 g for 30 min. The supernatant was collected and freeze dried, yielding the soy protein–soy polysaccharide Maillard reaction product (SPPMP). Above prepared SPP and SPPMP were sealed in bottles and stored at 4 °C until further use. 2.3. Preparation of citral-loaded emulsions stabilized by SPI, SPP and SPPMP The citral-loaded emulsions stabilized by SPI, SPP and SPPMP were prepared according to Yang et al. (2014). SPPMP-stabilized emulsions contained 10% (w/w) citral and 2.0%, 2.5%, 3.3% and 5.0% of SPPMP, respectively, which corresponded to the ratio of citral to SPPMP (w/w) at 5:1, 4:1, 3:1 and 2:1. Portions of the emulsions were adjusted to pH 7.0 or 3.0 with 2 M HCl with stirring at 250 rpm for 30 min. The citralloaded emulsions stabilized by SPP or SPI contained 10% citral (w/w) with the ratio of citral to SPP or SPI at 3:1 only. Triplicates were prepared for each emulsion. 2.4. Storage and heat treatment of emulsions Freshly made emulsions were stored in 20 mL capped amber glass vial with approximately 10 mL headspace in the dark at 25 °C. Some of the freshly made emulsions were heat-treated at 90 °C for 30 min
before storage, according to Xu and Yao (2009) to mimic a typical sterilization treatment in food processing.
2.5. Exposure to simulated gastrointestinal conditions Simulated gastric fluids (SGF) were prepared at pH 1.5, 2.0 and 2.5 containing 0.32% (w/w) pepsin and 0.2% (w/w) NaCl (Sarkar, Goh, Singh, & Singh, 2009). Simulated intestinal fluids (2×) were prepared at pH 7.5 containing 20 g/L pancreatin, 5 g/L bile salts, 80 mM K2HPO4, 150 mM NaCl and 40 mM CaCl2 according to Sarkar et al., with slight modifications (Sarkar, Horne, & Singh, 2010). Each freshly made emulsion was mixed with SGF (1:20, v/v) at 37 °C and divided into several aliquots stored in 10 mL amber glass tubes with continuous shaking at 100 rpm for 2 h. At each time point of 0, 0.5, 1.0, 1.5 and 2 h, three tubes from each treatment were removed from the incubation bath and the peptic digestion was stopped by adjusting to pH 7.5 with 2 M NaOH (Fafaungwithayakul et al., 2011), then cooled to 25 °C for GC analysis (after hexane extraction as described in Section 2.6). For the remaining tubes containing the emulsions after exposure to SGF, 2 M NaOH was added to adjust the pH to 7.5. Two models were adopted in the incubation under simulated intestinal conditions following the SGF incubation. In the first model, an equal volume of SIF (2 ×) was added to the emulsions after SGF incubations at pH 7.5, then incubated at 37 °C with shaking at ~ 100 rpm for 4 h. In the second model, the digesta from 12-day-old broilers (ileal digesta and jejunal digesta at a weight ratio of 1:1) was used instead of SIF at the same volume ratio, with the pH adjusted to 7.5. After incubation in simulated intestinal conditions for 4 h, samples were removed from the incubation bath, and cooled to 25 °C for GC analysis as described above.
2.6. Gas chromatography (GC) and gas chromatography–mass spectrometry (GC-MS) To measure the concentration of citral in an emulsion, approximately 2 mL of emulsion sample was mixed with hexane at 4 times the volume, and vortexing at speed of 1000 rpm for 30 min, then allowed to stand for another 30 min. The supernatant in each tube was collected and filtered through a syringe-driven filter unit (polytetrafluoroethylene, 0.22 μm), diluted to a citral concentration range of 20–100 μg/mL with hexane, and then analyzed by GC. An Agilent 6890 Series GC System equipped with a flame ionizing detector (FID) and a fused silica capillary column (30 m × 0.25 mm × 0.25 μm film thickness; temperature limits: 35–280 °C; SUPELCO WAX ™ 10) was used. The oven temperature was programmed as follows: initial temperature 60 °C (held for 1 min) followed by an increase at 8 °C/min to 240 °C and held for 1 min; injector and detector temperatures were 250 °C. Helium (2.0 mL/min) was used as the carrier gas. A sample (1.0 μL) was injected into the GC for testing and the two isomers of citral were identified by comparison to retention times with the analytical standard of citral from Sigma. The quantification of citral was conducted by determining the peak areas of neral and geranial, respectively, in the GC-FID graph and calculating against a standard curve made from the citral standard. Citral concentration was expressed as the sum of the two isomers. The degradation products of citral were identified by GC-MS. Electron impact (EI) mass spectrometric data were collected using a Hewlett–Packard 5973 mass spectrometer interfaced to a Hewlett– Packard 6890 gas chromatograph. The operating conditions of the mass spectrometer were as follows: split ratio 1:50, ionization voltage 70 eV and ion source temperature 140 °C. The operating conditions of the gas chromatograph were the same as in the GC-FID method. The mass spectrum of each identified compound was compared to the actual retention time and mass spectrum of each compound stored in the relevant standard databases (Wiley Registry® of Mass Spectral Data and NIST Mass Spectrometry Data Centre).
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100 Retention rate of citral (%)
2.7. Evaluation of citral stability The stability of citral in oil-in-water emulsions was evaluated by measuring the concentration of each of its isomers under different experimental conditions, and calculated as retention rate relative to day 0. Total retention rate of citral was calculated as the sum of the concentration of the two isomers at different experimental conditions relative to day 0. The day 0 concentrations were recorded as the concentration of citral isomers initially added to the emulsions. 2.8. Statistical analysis
3. Results and discussion 3.1. Chemical stability of citral in emulsions as influenced by citral to SPPMP ratio The chemical stability of citral was found to be dependent on the stability of SPPMP-stabilized emulsions, both of which varied with the citral to SPPMP ratio in the formulation. As shown in Table 1, increase of citral to SPPMP ratio resulted in an increase of average droplet size (Z-average) and polydispersity index (PDI) in the emulsions at the same pH. Increase of droplet size and polydispersity often associates with decrease in emulsion stability (Ray & Rousseau, 2013). At citral to SPPMP ratio of 4:1 and above, free citral was visible on the surface of the emulsion, indicating insufficient SPPMP to fully cover the citral droplets. As shown in Fig. 1, the retention rate of citral in SPPMPstabilized emulsions after 14-day-storage at neutral pH and 25 °C increased from 85.1% to 92.2% when the proportion of SPPMP was increased (from 5:1 citral:SPPMP to 2:1 citral:SPPMP). A similar trend was observed in the SPPMP-stabilized emulsions at pH 3.0 under the same storage conditions. Choi et al. (2010b) also reported that the retention rate of citral increased (from 25% to 54%) as the concentration of emulsifier (Tween 80) increased (from 0% to 5%) after one week storage at 20 °C and pH 3.0. The stability of citral was poorer, and retention rates were lower when emulsions were stored at pH 3.0 compared to pH 7.0 at all citral to SPPMP ratios. This observation is in agreement with the previous studies, in which the retention rate of citral was reduced as the pH decreased from 7.0 to 3.0 (Choi et al., 2009, 2010a; Mei et al., 2010). The difference between the retention rates of citral in SPPMP-stabilized emulsions at pH 3.0 and pH 7.0 became more apparent as the ratio of citral to SPPMP increased. Previous studies have demonstrated that citral inside emulsion droplets exhibited better chemical stability compared to free citral in the aqueous phase (Choi et al., 2009; Djordjevic et al., 2007, 2008); therefore, the chemical stability of citral in citral-loaded oil-in-water emulsions could be enhanced Table 1 The mean particle diameter and polydispersity index of citral-loaded emulsions with various ratios of citral to SPPMP after storage at 25 °C for 14 days.
7
3
a,b,c,d,e,f
Ratio
Mean particle diameter
(citral:SPPMP)
(Z-average, nm)
5:1 4:1 3:1 2:1 5:1 4:1 3:1 2:1
938 ± 44b 791 ± 25c 534 ± 14de 487 ± 9f 1064 ± 42a 825 ± 29c 561 ± 16d 528 ± 11e
Polydispersity index
0.47 ± 0.04a 0.32 ± 0.02b 0.21 ± 0.01d 0.18 ± 0.004e 0.45 ± 0.07a 0.36 ± 0.03b 0.24 ± 0.01c 0.21 ± 0.007d
Different letters in each column indicate significant difference (p ≤ 0.05).
90
pH=7.0
pH=3.0 c
bc
b
b
b
a
a
80
d
70 60 50 5:1
4:1
3:1
2:1
Ratio (citral: SPPMP) Fig. 1. Effects of citral to SPPMP ratio on the total retention rate of citral in SPPMP-stabilized emulsions after storage at pH 7.0 and 3.0 for 14 days at 25 °C. Different letters indicate significant difference (p ≤ 0.05).
with the improvement of emulsion stability. In a previous study, gum arabic (GA)-stabilized emulsions (5% GA, 10% citral, w/w) exhibited outstanding capacity to stabilize citral during storage, better than whey protein isolate (WPI)-stabilized emulsions (5% WPI, 10% citral, w/w) (Djordjevic et al., 2008). Compared with GA, SPPMP showed superior capacity for citral stabilization, especially at low pH. For example, after storage at 20 °C for 14 days, the retention rate of citral was 80% (pH 3.0) and 85% (pH 7.0) in the GA-stabilized emulsions (Djordjevic et al., 2008). Whereas, after storage for 14 days at a higher temperature (25 °C), the retention rate of citral was 92% (pH 3.0) and 90% (pH 7.0) in SPPMP-stabilized emulsions. As there was no significant difference (p N 0.05) between the stability of citral in emulsions with citral to SPPMP ratio at 3:1 and 2:1, the ratio of 3:1 was chosen for the rest of this study due to the higher citral content in the emulsion. 3.2. Citral stability in emulsions as influenced by storage time and heating The stability of citral in the SPI, SPP and SPPMP stabilized emulsions was very different (p ≤ 0.05), as indicated by the total retention rates of citral at each time point shown in Fig. 2. The SPPMP-stabilized emulsions showed superior ability to inhibit the loss of citral, yielding the highest retention rates at all tested time points in the storage period. The total retention rate of neral and geranial in GA-stabilized emulsions was about 80% when stored at pH 7.0 and 20 °C for 35 days, reported by Djordjevic et al. (2008), which is similar to our observed total retention
SPl
SPP
SPPMP
SPI (heating)
SPP (heating)
SPPMP (heating)
100 Retention rate of citral (%)
The statistical analysis was performed using Design-expert (Designexpert Version 9, Stat-Ease Int) software. Analysis of variance (ANOVA) was performed on the data, and a least significant difference (LSD) test with a confidence interval of 95% was used to compare the means.
pH of emulsion
359
75
50
25 0
30
60
90
120
150
Time (days) Fig. 2. Retention rate of citral in heated and unheated emulsions stabilized by SPI, SPP and SPPMP at pH 7.0 during storage at 25 °C.
Y. Yang et al. / Food Research International 69 (2015) 357–363
rate of neral and geranial in SPP-stabilized emulsion under similar storage conditions. When the SPPMP-stabilized emulsions were stored at higher temperature (25 °C), the total retention rate of neral and geranial was 85% after storage for 35 days and around 80% after storage for 70 days. In addition, SPPMP reduced the negative impact of heating on citral stability. In SPPMP-stabilized emulsions, a loss of 2.4 ± 0.8% of citral was found immediately after heating. In contrast, the loss rates of citral were found to be 5.0 ± 0.9% and 7.1 ± 1.6% for SPP- and SPIstabilized emulsions, respectively. It was also observed that the difference in retention rate between heated and unheated emulsions was significantly smaller for SPPMP-stabilized emulsions as compared to SPI- and SPP-stabilized emulsions in a prolonged storage period (p ≤ 0.05). After storage at 25 °C for 140 days, there were still 63.6 ± 2.3% and 69.3 ± 2.0% of citral remaining in the heated and unheated SPPMP-stabilized emulsions, respectively. The high stability of citral in the SPPMP emulsion could be attributed partially to the high stability of SPPMP-stabilized emulsions. It is widely acknowledged that the breakdown of emulsion and release of core material are usually associated with larger droplet size (Ray & Rousseau, 2013). The average particle size in SPPMP-stabilized emulsions was smaller than those in SPI-/SPP-stabilized emulsions during storage up to 140 days (p ≤ 0.05). In addition, the droplet sizes in the SPPMPstabilized emulsions did not change much after heat treatment and during the subsequent 2 months of storage at room temperature (Yang et al., 2014). The improved stability of citral by incorporation of SSPS into SPI-based emulsions could also be partially attributed to the reduction of negative surface charge and protein/amino acids content in the emulsions; the lowest negative surface charge and protein content were observed in the SPPMP-emulsions at neutral pH as compared to SPI- and SPP-emulsions (Yang et al., 2014). At neutral pH, ζ-potential of citral-loaded emulsion droplets stabilized by SPPMP was − 31.1 ± 0.5 mV, while those stabilized by SPP and SPI was − 37.6 ± 0.4 mV and − 46.2 ± 0.3 mV, respectively. The reduction of negative surface charge and protein/amino acid content in citral-loaded emulsions could slow the de-stabilization of citral. As mentioned earlier, polymers with high negative charges potentially attract more cations, such as iron ions, which could promote the oxidative transformation of citral (Choi et al., 2010a; Djordjevic et al., 2007; Hu, McClements, & Decker, 2003; Mei et al., 2010); whereas the presence of protein/amino acids at neutral pH could catalyze the de-acetylation of citral (Wolken et al., 2000). Several previous studies have also evaluated the capabilities of various emulsion systems to inhibit the transformation of citral, but few of them focused on long-term storage and the effect of heating, which are important factors to be considered for the applications of such systems. The results in the current study showed that the retention rate of citral was higher in SPPMP-stabilized emulsions during storage, compared to commonly used emulsifiers including sodium dodecyl sulfate, lauryl alginate, gum arabic, whey protein isolate, and soy protein isolate (Choi et al., 2010a; Djordjevic et al., 2007, 2008).
3.3. Exposure to simulated gastric and intestinal conditions 3.3.1. The stability of citral in simulated gastric conditions Maintaining the stability of citral at low pH is a major challenge for some applications, such as in intestinal delivery of citral as an antimicrobial agent and in acidic beverages as a flavoring and preservative agent (Maswal & Dar, 2014; Zhao et al., 2013). Although a number of investigations have been carried out on the stability of citral in acidic emulsions with various emulsifiers and/or antioxidants, none of them tested the stability of citral under gastric and intestinal conditions. The stomach is a major bioreactor for digestion of food, with strong acidity, proteolytic activity and vigorous agitation (Kong & Singh, 2010; Sarkar et al., 2009; Singh, Ye, & Horne, 2009). In this study, citral-loaded emulsions were exposed to SGF (0.32% pepsin, 0.2% NaCl, w/w, pH 2.0) with slow stirring at 37 °C, which mimics the environment of the stomach.
Because the retention rates of neral and geranial changed differently with time during incubation in SGF, they are expressed separately in Fig. 3. Of the three types of emulsions tested, the stability of citral in SGF was highest in SPPMP-emulsions. Previous studies have demonstrated that in an oil-in-water emulsion system, citral located outside the droplets was more susceptible to transformation, compared with that located inside the droplets (Choi et al., 2009, 2010a; Mei et al., 2010). In other words, the stability of citral in an oil-in-water emulsion was inversely associated with the release of citral from emulsion droplets: higher release rate of citral to the aqueous phase leads to lower stability of citral. The observations from the current study and our previous study (Yang et al., 2014) are in agreement with this conclusion. In our previous study, the release rate of citral was found to be the lowest in SPPMP-emulsions compared with SPI- and SPP-emulsions. In general, neral was less stable than geranial in SGF. However, the difference in retention rates between neral and geranial was not significant in SPPMP-emulsion after 2 h incubation in SGF. In contrast, the differences were found to be significant for SPI- and SPP-emulsions after 30 and 60 min incubation in SGF, respectively (p ≤ 0.05); the observed difference increased with incubation time. This result agrees well with an earlier report that the transformation of neral was faster than geranial (p ≤ 0.05) in gum arabic-stabilized emulsions during storage under acidic conditions (pH 3.0) (Djordjevic et al., 2008). In acyclic systems, trans isomers are usually more stable than cis isomers, which is due to increased unfavorable steric interactions in the cis isomer (March, 1992). Fig. 4a shows a GC chromatogram of citral before emulsification; Fig. 4b shows a GC chromatogram of citral and its transformation products in emulsions stabilized by SPI, SPP and SPPMP after SGF incubation. In comparison with Fig. 4a, two new peaks appeared in Fig. 4b at retention times of 11.5 min and 12.4 min, respectively. These were identified by GC-MS analysis as p-mentha-dien-4-ol and p-mentha-diene-8ol, the major products from the first stage of acid-catalyzed transformation of citral (Kimura et al., 1982; Peacock & Kuneman, 1985). Much smaller peaks at retention times of 13.4 min and 14.6 min (data not shown) were identified as the further oxidation products of pmentha-dien-4-ol and p-mentha-diene-8-ol: -p-dimethylstyrene and p-methylacetophenone (second-stage transformation products of citral) respectively (Kimura et al., 1983b; Peacock & Kuneman, 1985; Schieberle & Grosch, 1988). These oxidation products were also previously identified as the main transformation products of citral in citralcontaining solutions or citral-loaded emulsions stored at 25–40 °C for 7–40 days at pH 3.0. (Liang, Wang, Simon, & Ho, 2004; Maswal & Dar, 2013; Ueno et al., 2004; Yang et al., 2011b; Zhao et al., 2013). It was
SPPMP geranial SPPMP neral
SPP geranial SPP neral
SPI geranial SPI neral
100 Retention rate of citral (%)
360
90
* * *
80
* * *
70
*
60 0
30
60
90
120
Time (min) Fig. 3. Retention rate of neral and geranial in emulsions stabilized by SPI, SPP and SPPMP during incubation in SGF at pH 2.0. *indicates significant difference between geranial and neral in the same emulsions at the same time point (p ≤ 0.05).
Y. Yang et al. / Food Research International 69 (2015) 357–363
a
361
100 120
Neral
Retention rate of citral˄%˅
FID response (PA)
100 80 60 40 20 0 10.5
11
11.5
12
12.5
13
Neral (heating)
Geranial
e
90
de
d
a
cd
cd
c
80
Geranial (heating)
de cd
ac a
b
70
13.5
Retention time (min)
60 1.5
b
2.0
2.5
pH 100 SPPMP SPP SPI
FID response (PA)
80
Fig. 5. Retention rates of neral and geranial in heated and unheated SPPMP-stabilized emulsions after incubation in SGF at different pH for 2 h. Different letters indicate significant difference (p ≤ 0.05).
60
40
20
0 10.5
11
11.5
12
12.5
13
13.5
Retention time (min)
Fig. 4. Gas chromatogram: (a) citral before emulsification; (b) citral and its transformation products in emulsions stabilized by SPI, SPP and SPPMP after SGF incubation at pH 2.0 for 2 h.
reported that while the retention rate of citral decreased from 80% to 0%, the concentration of -p-dimethylstyrene and p-methylacetophenone (second-stage transformation products of citral) increased by 6-fold (Liang et al., 2004). In contrast to the previous studies, the transformations of citral after SGF incubation at pH 2.0 mostly remained at the first stage in the current study, due to the short incubation time (2 h) and the protection provided by the emulsion. The concentrations of pmentha-dien-4-ol, p-mentha-diene-8-ol and their oxidation products were lowest in the SPPMP-stabilized emulsions, compared with SPIand SPP-stabilized emulsions (Fig. 4b). This result suggests that SPPMP-stabilized emulsions could effectively retard the transformation of citral in simulated gastric conditions. 3.3.2. The influence of pH of SGF and pre-heating of emulsions For live animals, the pH of the stomach fluids varies from pH 1 to 3 depending on the digestive status (i.e. fullness, appetite). In this study, SPPMP-stabilized emulsions (heated and unheated) were tested in SGF at pH 1.5, 2.0 and 2.5. The stability of the citral in unheated SPPMP-stabilized emulsions was found to be responsive to the pH of SGF (Fig. 5). The retention rate (p ≤ 0.05) of geranial decreased significantly as the pH values decreased, whereas for neral, the retention rate did not significantly change as the pH decreased from 2.5 to 2.0. The retention rate between neral and geranial showed significant differences only as the pH decreased to 1.5 (p ≤ 0.05). The higher concentration of H+ found at lower pH in an emulsion system would be the main factor that is responsible for the transformation of citral because H+ participates in the acid-induced transformation of citral to form monoterpene alcohols (Kimura et al., 1982). This was confirmed by more rapid transformation of citral in an emulsion system at pH 3.0, compared with that at pH 7.0 by Yang, Tian, Ho, and Huang (2011a). Although the retention rate of citral also decreased in SPPMP-stabilized
emulsions as pH decreased, the extent was limited. The retention rate of citral in SPPMP-stabilized emulsions after SGF incubation at pH 1.5 was calculated to be 10% higher than the retention rate of citral in SPP-stabilized emulsions at the same pH (data not shown), and 4% higher than the retention rate of citral in SPP-stabilized emulsions at pH 2.0 (data from Figs. 5 and 3). Fig. 5 also shows the influence of heat treatment on the stability of citral in SPPMP-stabilized emulsions during SGF incubation. The difference in the retention rate of citral between heated and unheated emulsions during SGF incubation was somewhat dependent on the pH of SGF; the difference in retention rates of citral (neral and geranial) between heated and unheated SPPMP-stabilized emulsions was only significant at pH 1.50 (p ≤ 0.05). In contrast, for SPI- or SPP-stabilized emulsions, the retention rates of citral in the heated emulsions were significantly decreased, compared to the unheated emulsions at pH 1.5, 2.0 and 2.5 during SGF incubation (data not shown). The stability of citral in an emulsion system during SGF incubation would be related to the pepsin-digestibility of the emulsifiers. The pepsin-digestibility of SPI was shown to increase after heat treatment (Tsumura, Saito, Kugimiya, & Inouye, 2004). In heat-treated emulsions, the increase of pepsin-digestibility of SPI could result in disintegration of emulsion droplets and the release of citral during SGF incubation. On the contrary, pepsin-digestibility of SPPMP might be decreased, as the SPPMPstabilized emulsion exhibited enhanced stability under gastric conditions and better resistance to heat treatment (Yang et al., 2014). Therefore, the improved chemical stability of citral in a SPPMP-stabilized emulsion can be attributed mainly to the enhanced stability of the emulsion system. 3.3.3. Exposure to simulated gastro-intestinal tract Two in vitro models were chosen for the current study. In the first model, the emulsions were exposed to simulated intestinal fluid (SIF) after 2 h SGF incubation; in the second model, the intestinal digesta collected from 12-day-old chicks was used instead of SIF. The total retention rates of citral in SPPMP-stabilized and SPPstabilized emulsions were about 1.5 and 1.3 times higher, respectively, than that in SPI-stabilized emulsions after incubation in simulated gastrointestinal fluids (Fig. 6). Consequently, the concentration of citral transformation products was highest in SPI-stabilized emulsions and lowest in SPPMP-stabilized emulsions (Fig. 7). Compared with the corresponding emulsion after incubation in SGF only (Fig. 4b), the concentration of citral transformation products increased after SGF-SIF incubation (Fig. 7). A similar trend was observed for the emulsions incubated with chicken digesta as shown in Fig. 6, although the
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90 Retention rate of citral (%)
SIF
4. Conclusions
Digesta
80
Incorporation of SSPS could enhance the stability of citral in SPIbased emulsion systems. Compared with soy protein–polysaccharides mixture (SPP), soy protein–polysaccharide Maillard reaction products (SPPMP) offered significantly better protection to citral in a prolonged storage period, upon heat treatment, and in simulated gastrointestinal tracts. The high emulsification ability and stability of SPPMP helped to protect citral from interaction with compounds in the aqueous phase and other environmental factors that could induce the transformation of citral and cause loss of its functionality. The results from the present study suggest that the SPPMP-stabilized emulsion system could be useful for the protection and targeted delivery of citral to the small intestine.
c
70
b b
60 a a
50
d
40 30 SPI
SPP
SPPMP
Fig. 6. Retention rate of citral in emulsions stabilized by SPI, SPP and SPPMP after incubation in simulated gastro-intestinal conditions with SIF or digesta. Different letters indicate significant difference (p ≤ 0.05).
corresponding retention rates were significantly lower compared to those in SIF. Further loss of citral in SIF and the digesta is mainly due to the breakdown of emulsions and subsequent digestion (Yang et al., 2014). Soy proteins are subject to hydrolysis by proteases present in the SIF and the digesta, which may lead to disintegration of emulsion droplets. Incorporation of SSPS to SPI, either by physical interactions or covalent-links, may spatially shield peptide bonds against proteolysis, thus retard the release of citral from the droplets. Our previous study (Yang et al., 2014) confirmed that the release rate of citral from SPPMP-stabilized emulsions was significantly slower during exposure to SIF, than that from SPI- and SPP-stabilized emulsions. The current results indicate that the SPPMP-stabilized emulsion was the most effective in preventing the loss of citral under simulated gastrointestinal conditions among the three emulsions tested. It is not surprising that the retention rate of citral was lower in chicken digesta than that in SIF. It is well known that there are many kinds of chemical compounds in the digesta. Among those, metal cations, amino acids and other oxidants could interact with the released citral, inducing its transformation. Other macromolecules and small molecules could influence the stability of citral by different mechanisms. In addition, the concentration and locations of citral transformation products may also impact the rate and extent of further transformation of citral in emulsion systems (Choi et al., 2010a). More studies are needed to investigate the effects of different types of components present in the gastrointestinal tract on the stability of citral and citral-loaded emulsions.
100 SPPMP SPP SPI
FID response (PA)
80
60
40
20
0 10.5
11
11.5
12
12.5
13
13.5
Retention time (min)
Fig. 7. Gas chromatograms of citral and its transformation products in emulsions stabilized by SPI, SPP and SPPMP after SGF incubation at pH 2.0 for 2 h followed by SIF incubation at pH 7.5 for 4 h.
Acknowledgments This research is supported by Agriculture and Agri-Food Canada and Canadian Poultry Research Council under the Growing Forward Program (RBPI# 1896 and #13-1039). We also gratefully acknowledge the financial support received from Doctor Candidate Foundation of Jiangnan University (JUDCF10061). Yuexi Yang is a visiting Ph. D. student funded by China Scholarship Council through the AAFC-MOE Program. We wish to thank Ms. Linda Lissemore of the University of Guelph for conducting the GC-MS analysis and Ms. Marta Hernandez of AAFC for helping with GC analysis.
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