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JFS: Vol 76, Number Industrial Application Briefs Industrial Applications of Selected JFS Articles We are seeing just how important flavor compounds are—the amount of pungency in onions, the characterization of flavor in certain kinds of honey As standardization of flavor becomes key, products can even be in trouble for tasting better than they should! Instrumentation helps—the human tongue and nose is the final judge, but numbers can help More papers are appearing with work on nanotechnology, as well; an understanding of how this new science works is imperative to the up-to-snuff technologist (and engineer and marketer) Learning a Little More about Nanoemulsion Phases A paper titled “Optimization of β-Casein Stabilized Nanoemulsions Using Experimental Mixture Design” outlines the changes in viscosity and glass transition temperature that exists in the continuous phase of nanoemulsion systems, and how those attributes affected stability of a product Emulsions were made that included β-casein at levels, lactose and trehalose at several levels Higher levels of β-casein content resulted in increased viscosity and decreases in melting temperatures A mixture design was used to predict the optimum levels of lactose and trehalose needed to reach minimum and maximum Tg and viscosity in solution at fixed protein contents C1108–1117 Keeping Probiotics Useful standard test since 1961, measuring the pyruvic acid concentration in onion juice However, researchers found that the absorbency of the color adduct of the reaction dropped fast, as compared to that of the pyruvic acid standards, which meant that the pyruvic acid was higher than estimated Said the researchers, “by measuring the absorbency at min, we have demonstrated that accuracy could be substantially improved.” The results were published in “Underestimation of Pyruvic Acid Concentrations by Fructose and Cysteine in 2,4-Dinitrophenylhydrazine-Mediated Onion Pungency Test.” Alliinase action in juice (fresh or cooked) and bulb colors did not affect the degradation Some organic acids indigenously found in onion, such as ascorbic acid, proline, and glutamic acid, did not reduce the absorbency However, fructose within the onion juice or supplemented caused the degradation of the color adduct, whereas sucrose and glucose also affected the test, but had a lesser effect Degradation rates increased proportionally as fructose concentrations increased up to 70 mg/mL Cysteine was found to degrade the pyruvic acid itself before the pyruvic acid could react with DNPH This happened rapidly So if the degree of pungency of onion ingredients is important to control, this study is important C1136–1142 Films for Packaging Are Built with Agar, Special Clays Water vapor adsorption behaviors are changed in nanofilms made from agar and nanoclay according to the type of nanoclays used In the paper titled “Water Vapor Adsorption Isotherms of Agar-Based Nanocomposite Films”, the author discusses methods used to get an understanding of the effects of various nanoclays: The Guggenheim–Anderson–de Boer (GAB) isotherm model parameters were estimated by using both polynomial regression and nonlinear regression methods, and the behavior of the nanocomposite films was found to be greatly influenced by the type of clay It was found that the GAB model fitted adequately for describing experimental adsorption isotherm data for the film samples The monolayer moisture content (mo) of the film samples was also greatly affected by the type of nanoclay used, that is, mo of nanocomposite films was significantly lower than that of the neat agar film For background information, for those not into nanotechnology, bionanocomposites are hybrid materials of biopolymer with inorganic fillers which have at least one dimension in the nanometer scale It is generally known that nanoscale dispersion of the filler phase in the polymer matrix leads to property enhancements such as decreased permeability to gases (O2 , How Pungent Is that Onion? CO2 , and water vapor) and liquids, better resistance to solvents, In determining how pungent an onion is, one standard reacts increased thermal stability, and improved mechanical properties onion juice with 2,4-dinitrophenylhydrazine (DNPH) It’s been a N68–72 Freeze-drying probiotics and storing the dried powder, the usual way to process the probiotics for commercial use, appears to reduce the viability of the materials A group of researchers are looking into the efficacy of using a glassy state for the little varmints, and retaining the glassy state during drying In “Role of Glassy State on Stabilities of Freeze-Dried Probiotics” the authors describe the glassy state that should be retained during freezing, drying, and storage of cells Insight into the role of glassy state has been largely adopted from studies conducted with proteins and foods, especially sugars Say the researchers, “Current understanding of the role of the glassy state on viability of probiotics is not only valuable for the production of fermented foods and nutraceuticals but also for the development of nonfermented functional foods that use the dried powder as an adjunct.” The paper, a review of recent findings regarding glassy states and the effects on probiotic survival, notes that 1000 articles and reviews were published in 2008, and more information is appearing, supporting the introduction of $16 billion in the U.S alone in 2008 R152–156 v JFS: Vol 76, Number Editorial Speaking of Word Count About a year ago, we informed the potential authors of future manuscripts submitted to sections of the Journal of Food Science that we would be limiting word count on manuscripts In this action, we were trying to decrease the number of printed pages particularly for JFS while enhancing the readability of the papers In a survey of printed papers at that time, Amanda Ferguson discovered that most manuscripts to JFS were a little over 5,000 in word count, and manuscripts in Comprehensive Reviews in Food Science and Food Safety were all over 10,000 words With those data as the guideline, we set a JFS research manuscript word count limit (excluding tables and figures) at 5,000 words and minimum word count for CRFSFS at 10,000 words Starting November 2011, authors were asked to voluntarily submit word count on manuscripts and in January 2011, words count was required Needless to say, we had some flexibility in applying the word count rule On the other hand, I received not one letter to the editor decrying the use of word count or complaining that it was too limiting Nor did I receive any oral argument while I attended the IFT AMFE in June I take this inaction as a sign of not only acceptance but of support for word count limitation With that in mind, I asked Amanda to again a survey of word count on our accepted manuscripts The data for a sampling of papers published in 2011 are in and they tell the following story Word Counts 2011 JFSE Avg CRFSFS Avg JFS-Concise Reviews Avg JFS-Research Sections Avg vi Mean Range 3014.25 15219.75 7893.67 4544.28 922-6312 10000-21550 5266-12606 1930-8454 Of the 141 papers in the JFS research sections, only 18 were over the 5,000 word limit, and only were more than 1000 words over The mean of 4500 word count for the research sections of JFS compares to the October 2010 reported mean of 5140 and range of 2350-8560 Only review paper in JFS was over the 10,000 word limit The effect of the decrease in word count was to reduce the page count per paper from 7.3 to 7.0 In other words, the change achieved the result we were hoping for and there were not untoward consequences Now, for the kicker, should we be looking at a 4,500 word count limit for research papers submitted to JFS for 2012? Send your thoughts to me at dlund@cals.wisc.edu Daryl Lund Editor in Chief R: Concise Reviews in Food Science Role of Glassy State on Stabilities of Freeze-Dried Probiotics Chalat Santivarangkna, Mathias Aschenbrenner, Ulrich Kulozik, and Petra Foerst Abstract: High viability of dried probiotics is of great importance for immediate recovery of activity in fermented foods and for health-promoting effects of nutraceuticals The conventional process for the production of dried probiotics is freeze-drying However, loss of viability occurs during the drying and storage of the dried powder It is believed that achieving the “glassy state” is necessary for survival, and the glassy state should be retained during freezing, drying, and storage of cells Insight into the role of glassy state has been largely adopted from studies conducted with proteins and foods However, studies on the role of glassy state particularly with probiotic cells are on the increase, and both common and explicit findings have been reported Current understanding of the role of the glassy state on viability of probiotics is not only valuable for the production of fermented foods and nutraceuticals but also for the development of nonfermented functional foods that use the dried powder as an adjunct Therefore, the aim of this review is to bring together recent findings on the role of glassy state on survival of probiotics during each step of production and storage The prevailing state of knowledge and recent finding are discussed The major gaps of knowledge have been identified and the perspective of ongoing and future research is addressed Keywords: freeze drying, glass transition, lactic acid bacteria, probiotics Introduction Research and interest regarding probiotics is receiving more focus than ever before For example, in 2008, more than 1000 articles and reviews were published on the subject and more than 2000 probiotic products launched (Jankovic and others 2010) The global market for probiotic products was worth US$16 billion in 2008, and the estimated target is a total of US$19.6 billion in sales in 2013 (Granato and others 2010) For food applications, probiotics are mainly employed alone or together with starter cultures in fermented dairy products (for example, yogurts and cultured drinks) In addition, probiotics are also potentially used as nutraceuticals and dry adjunct in nonfermented and nondairy products, such as fruit juices, cereals, dried powder foods (RiveraEspinoza and Gallardo-Navarro 2010) In either case, high viability of probiotics is of great importance in order to ensure immediate recovery of fermentation activity (especially Direct Vat Inoculation-DVI cultures) and to meet the minimal requirements for health-promoting effects The standard process for the production of dry probiotics is freeze-drying The typical freeze-drying process consists of steps: freezing, primary drying, and secondary drying During these steps, cells are exposed to various stresses, especially dehydration, compromising cell survival A further survival reduction occurs during storage of the dried powder, where the key storage conditions such as relative humidity and temperature play a major role In addition to physiological states of cells, the physical state of the surrounding sample matrix is believed to be critical for survival of probiotics The high viscosity of a surrounding amorphous glass can inhibit diffusion and slow down deleterious reactions or changes in the structures and chemical composition The role of glassy state on the functionalities, bioactivity, and stability of enzymes, pharmaceutical proteins, and foods has been widely reported Nevertheless, current studies on the role of glassy state, particularly with probiotics, are on the increase and both common and explicit findings have been found It is an aim of this review to bring together recent findings on the role of glassy state on survival of probiotics during each step of the production and of the storage period The prevailing state of knowledge related to the role is revisited, and recent finding is discussed Glassy State The glassy state is referred to an amorphous metastable state that resembles a solid but without any long-range lattice order, that is, the position of molecules relative to another is more random In other words, it has a solid characteristic/appearance but a molecular arrangement that is more typical for liquids A glass has an extremely high viscosity (for example, typically ≥ 1012 Pa s) and shows temperature-dependent transition (Slade and Levine 1991) When a glass is heated above a certain temperature, the molecules gain translational mobility and enter a liquid-like state The transformation of solid- to liquid-like state is known as glass transition The most common parameter describing the glassy state and its transition is the glass transition temperature (Tg or Tg for a maximally freeze-concentrated solution), below which materials exhibit the extremely high viscosity At T < Tg , diffusion limited deterioration reactions are inhibited because water in the amorphous glass is immobilized and unavailable In various studies, sugars or strong glass-forming polymers have MS 20110302 Submitted 3/9/2011, Accepted 6/21/2011 Authors are with Chair been added in an effort to increase T of dried probiotics This g for Food Process Engineering and Dairy Technology, Centre of Life and Food Sciences, Technische Univ Măunchen, 85354-Freising, Germany Direct inquiries to author is based on the observation that many anhydrobiotes accumulate large amount of sugars, especially trehalose and sucrose, inside the Foerst (E-mail: petra.foerst@wzw.tum.de) cells, for example, 25% of DW (Buitink and Leprince 2004) The R152 Journal of Food Science r Vol 76, Nr 8, 2011 C 2011 Institute of Food Technologists R doi: 10.1111/j.1750-3841.2011.02347.x Further reproduction without permission is prohibited presence of sugars on both sides of the cells was considered being necessary for protection against freezing (Duong and others 2006) and dehydration (Tymczyszyn and others 2007) Crowe and others (1987) found that intra- and extracellular sugars provide protection of proteins and membranes by forming a glassy matrix that is able to interact via hydrogen bonding This protective effect of sugars on biomolecules was reported to appear only in case of amorphous carbohydrate glasses, but not in case of crystalline sugars (Pikal and Rigsbee 1997; Izutsu and Kojima 2002) In contrast to the drying of pharmaceutically relevant proteins, where the sensitive biomolecules are directly embedded in the amorphous sugar glass, several drying-sensitive components of bacterial cells are situated in the intracellular space Therefore, the protectant must be transported through the cell membrane Thus, many high molecular weight (MW) polymers that show limited transportation into cells or in penetration and interaction with the phospholipid headgroups of the cell membrane are not very effective in protecting dried probiotics (Semyonov and others 2010) According to Potts (1994), it is still not clear if bacterial glasses exist The differential scanning calorimetry (DSC) technique which is commonly used for the detection of glass transition, is insensitive for cells, where changes in heat capacity are small and or when many transitions occur in the same temperature range For example, Fonseca and others (2001) showed that the Tg of washed cells of Lactobacillus bulgaricus cannot be detected Furthermore, Cerrutti and others (2000) and Fonseca and others (2001) indicated that the reported Tg is mainly obtained from the added solutes or the drying matrix However, Hoekstra and others (2001) and Sun and Leopold (1997) showed that the state of the cytoplasm is considered to be of crucial importance for the dehydration tolerance in organisms Thus, the glassy state of dried cells (if there is any) needs to be considered carefully concentrated matrix with entrapped microbial cells is essential for the survival during freezing (Pehkonen and others 2008) Given Tg of skim milk (–50 ◦ C), sucrose (–46 ◦ C), and trehalose (–40 ◦ C), which are the most common suspending medium used in many studies, and Tg of pure water (–135 ◦ C), the maximally freezeconcentrated matrix should be obtained by common industrial freezing protocols, for example, immersion in liquid N2 (–196 ◦ C) Otherwise, the freezing temperature should be selected with care to ensure a maximally freeze-concentrated state and formation of a glassy structure of the unfrozen continuous phase of the cell suspension In freeze-drying formulations, cryoprotectants are used to forestall cell injury Some viscous cryo-protectants (for example, glycerol, sugars, and polymers) increase the viscosity of the freezeconcentrated solution or cytoplasm depending on their permeability Therefore, the glassy state can be reached at a lower cooling rate and a higher freezing temperature (Morris and others 2006) The annealing step is commonly used for the freeze-drying of proteins and pharmaceuticals However, it is not applied for the production of freeze-dried probiotics Annealing is a process, where frozen samples are kept isothermally at a temperature between Tg and the onset of melting temperature During annealing, freezable water entrapped in amorphous regions (due to rapid freezing) is crystallized into larger and more uniform ice crystals This is especially true for the frozen probiotics, which are commonly produced by rapid freezing using liquid nitrogen A pellet of mm diameter is cooled from to –50 ◦ C in approximately 10 s or at a freezing rate of approximately 300 ◦ C/min (Oetjen and Haseley 2003) The increased content and uniform ice formation improve the efficacy of subsequent drying process The knowledge of the influence of annealing on probiotics’ survival and stability is very limited In a study by Ekdawi-Sever and others (2003), it was reported that the annealing does not cause cell death, but improves storage stability and reduces browning reaction of L acidophilus Role of Glassy State during Freezing The typical freeze-drying or lyophilization process consists of steps: freezing, primary drying or sublimation, and secondary drying or desorption The ice crystals formed during freezing determine the morphology and distribution of the pores (macroscopic cake structure) that are formed during the removal of ice crystals Thus, the freezing process significantly determines the drying behavior of the sample throughout the following process steps In order to ensure rapid drying and to facilitate vapor migration during drying, ice crystals in the suspension should be large and contiguous On the one hand, large ice crystals can only be achieved by relatively slow freezing rates (not with liq N2 ) On the other hand, these large ice crystals can induce cell damage due to mechanical stress Generally, slow freezing may accompany the eutectic crystallization of buffer salt components, which is also suspected to cause membrane damage (Morgan and others 2006) In other words, optimum freezing conditions often require a compromise between the requirements of the bacteria and the drying performance For the production of probiotics, frozen granules are commonly produced by distributing the cell suspension through a droplet disc with pores or through a nozzle into liquid N2 In addition to direct freezing in liquid N2 , innovative rapid freezing methods were explored by Volkert and others (2008) The frozen granules are produced by the spray freezing, where cell suspension is sprayed in an air blast freezer to get droplets (for example, size of ca to 30 μm) For a frozen solution, at Tg ,the high viscosity of the unfrozen phase will inhibit ice crystal formation; and therefore, a maximally freeze-concentrated solution is formed (Roos 1997) It has been suggested that the formation of a maximally freeze- Role of Glassy State during Drying It is the current opinion that the product temperature during removal of ice crystals should not be higher than the critical temperature This critical temperature commonly refers to Tg (or Tg after sublimation) In this respect, the processing conditions, (for example, pressure and shelf temperature during sublimation) must be controlled so that the product temperature is not higher than Tg during drying For this reason, solutes with high Tg are often added to increase Tg so that the drying can be carried out at a higher temperature, and a high Tg of final dried products is obtained to provide a high storage stability Disaccharide sugars and oligomeric sugars are preferred as additives for freeze-drying not only because they exhibit a higher Tg (Adams and Ramsay 1996), but also because they can be easily vitrified (Franks 1998; Ward and others 1999) For sugar alcohols such as mannitol, caution must be exercised Mannitol can easily separate from a frozen solution in the form of a crystalline phase (Adams and Ramsay 1996; Kim and others 1998), resulting in a loss of the product stability after freeze-drying (Izutsu and others 1994; Izutsu and Kojima 2002) This reason has been proposed for the little or no protection conferred by mannitol on freeze-dried malolactic cultures (Zhao and Zhang 2005) Recently, “collapse temperature” which is defined as the maximum temperature preventing the structure of the dried product from macroscopic collapse (Tc ) (Fonseca and others 2004a) was proposed to use as the critical temperature for freeze-drying of drying formulation with cells In comparison, Tc and Tg are measured with techniques based on different principles DSC has been Vol 76, Nr 8, 2011 r Journal of Food Science R153 R: Concise Reviews in Food Science Glassy state of dried probiotics R: Concise Reviews in Food Science Glassy state of dried probiotics commonly used over decades to determine the Tg as a mid- or onset point of the temperature range that the endothermic shift in heat capacity appears The measurement of Tg is carried out with a representative frozen sample ex situ at atmospheric pressure In contrast, Tc is commonly determined by a freeze-drying microscope as the visual structural collapse during the simulated sublimation of ice crystals under a set vacuum level In other words, Tc reflects the physical state of frozen matrix during drying, whereas Tg rather reflects the physical state regardless of the drying conditions The state diagram showing glass transition temperature and collapse temperature is depicted in Figure (adapted and modified from Roos 2010) In the absence of cells, the difference between Tg and Tc is very small Although the presence of cells does not clearly influence Tg (Fonseca and others 2001; Schoug and others 2006), it significantly increases Tc (Fonseca and others 2004a) Bacteria can give some kind of structure and thus reduce or avoid viscous flow when Tg of pure sugar solution is reached The increase depends on the cell types, that is, size, shapes, and cell chain formation and concentration (Fonseca and others 2004b) As a result, a freeze-drying matrix with cells is more robust, and when Tc is taken as the critical temperature, it allows the drying stage with a higher product temperature (or practically drying temperature) This is of economical importance because it is estimated that the increase in a degree of product temperature will decrease primary drying time by about 13% (Tang and Pikal 2004) Nevertheless, the opinion that cells should be retained in glassy state during drying lacks clear empirical evidence The physical state has been measured mostly by analyzing the frozen and dry sample before and after drying, while the physical state during drying is changed with drying time and conditions In our recent study (Foerst and others 2010), several drying protocols were carried out, and the viability was considered in relation to physical state, residence time in rubbery state and moisture content of samples The study showed that glassy state may not play a significant role on viability of cells during drying step of freeze-drying The survival decreased similarly for conditions where the samples remained in glassy state for the whole part of drying, and for con- dition where the difference between product and glass transition temperature was so high that the sample showed a collapsed structure The results are in agreement with unexpected results from a recent study by Schersch and others (2010) with pharmaceutically relevant proteins Loss of biological activities of the proteins was not observed in collapsed and noncollapsed cakes The collapsed lyophilizate, the appearance of which is unacceptable for pharmaceutial products, is not critically important for probiotic products The dried lyophilizate has to be milled into powder to be mixed with other food components, mixed with other fillers for probiotic tablets or capsules, as well as used as starter cultures When the collapsed structure as a result of drying does not negatively affect the viability of cells, the proposed concept “collapse drying,” which is expected to substantially reduce drying time (Schersch and others 2010) can be possibly applied for the production of freeze-dried probiotics In addition to free water, which is removed during primary drying, cells contain ca 0.25 g water/g dry weight of strongly associated water that is unfreezable This strongly associated water is removed by desorption The shelf temperature is elevated to promote desorption of water At the end of the secondary drying, the sample should have a water content that is optimal for storage It has been suggested that the moisture content of 1% or less is required for a long-term shelf life (Nakamura 1996); however, Gardiner and others (2000) showed that moisture removal below 4% may be considered practically enough It was also reported in a study by Zayed and Roos (2004) that the optimal moisture content for storage of freeze-dried L salivarius ssp salivarius is in the range of 2.8% to 5.6% Therefore, it is still debatable whether cells should be dried to moisture content as low as possible An argument is that lipid oxidation is enhanced at very low water contents, and water acts as a protectant against oxidation Role of Glassy State during Storage Low moisture contents of dried cells after drying not guarantee high stability during storage The moisture is not constant, and Figure 1–State diagram showing different regions and state of a drying matrix: Tm , onset of ice melting; Tm , onset of ice melting in a maximally freeze-concentrated solute matrix; Tg , glass transition temperature; Tg , glass transition temperature of the maximally freeze-concentrated solute matrix; Tgw , glass transition of water, and Tc , collapsed temperature Tc approximate to Tg and increases in the presence of cells The shaded area shows a temperature range for maximum ice formation for the solute concentration Cg The glass line indicates the glass transition temperature at various solute concentrations (adapted and modified from Roos 2010) R154 Journal of Food Science r Vol 76, Nr 8, 2011 desorption, adsorption, and the glass transition may occur during storage depending on the specific combination of atmospheric relative humidity and storage temperature Equilibrium of ambient conditions may only take a few hours or days (Kurtmann and others 2009b) or as long as to to mo (Fonseca and others 2001) This equilibration period is often not taken into account in studies with the storage stability and Tg At a critical storage temperature Tg , the critical water content and storage relative humidity (or aw ) can be determined when information on sorption isotherm of cells is available (Figure adapted and modified from Fonseca and others 2001) Information on the sorption isotherm of dried probiotics is necessary to avoid inactivation due to moisture gain during storage Furthermore, it can be used to correlate the storage relative humidity and the final moisture content of dried cells to avoid unnecessary drying time from drying of cells far beyond the moisture content corresponding to storage aw However, in comparison to foods, very little information on sorption isotherm of probiotics is available Notably, stability of probiotics is commonly considered with respect to survival The role of glassy state has never been studied in correlation to probiotic properties, although the properties are vital for probiotic products and there is a report about change in the properties during storage The amount of bacteriocin production in freeze-dried vaginal lactobacillus strains was significantly reduced after storage for 12 mo, whereas the production of other antimicrobial substances (lactic acid and hydrogenperoxide) and the auto-aggregation were still retained (Juarez Tomas and others 2009) Nevertheless, recent studies on storage of dried probiotics cast doubt upon the hypothesis that chemical reactions are completely halted in the glassy state It does not solely determine the storage stability of dried cells, and inactivation still occurs during storage at a temperature T – Tg = (Higl and others 2007; Kurtmann and others 2009b) It was suggested that in order to achieve nearly complete reduction of molecular movements, amorphous pharmaceutical solids should be stored 50 ◦ C below Tg (Hancock and others 1995) Similar findings were reported also in a study with L coryniformis by Schoug and others (2010) and with L rhamnosus by Miao and others (2008) where viability of cells clearly decreased at T – Tg higher than –50 ◦ C and ca –30 ◦ C, respectively Furthermore, the glassy state does not play major protective role against major storage deteriorative reactions such as lipid oxidation (Andersen and others 1999; Kurtmann and others 2009a), because the free radical reaction is not diffusion limited In addition, lipid carbonyls as formed as secondary oxidation products from lipid oxidation have been suggested to participate in browning reactions through reactions with amino groups in Maillard-like reactions Browning reaction is related to viability loss in freeze-dried cells (Kurtmann and others 2009c) It was suggested also that the nature of the sugar was more important for storage stability than the physical state of the matrix with the nonreducing sucrose providing better stability than the reducing lactose (Kurtmann and others 2009b) Alternatively, it was also proposed that a biomaterial is most stable at or below its monolayer moisture content or aw , which varies in each biomaterial and with environmental conditions (Pitombo and others 1994) For example, the monolayer moisture content of L bulgaricus (Fonseca and others 2001) and encapsulated L rhamnosus (Ying and others 2010) was 10% and 3%, respectively This concept cannot completely explain the storage stability of dried cells as well, considering that an increase in temperature significantly affected the inactivation rate but minimally affected the water activity Moreover, it provides only information on strongly associated and free water but does not indicate when the molecular mobility starts to increase A strong combined effect of water activity and temperature was reported and it was suggested that aw and Tg are coupled (Kurtmann and others 2009b) Unlike freeze-dried proteins, a bacterial cell is not a unique entity but contains the outside compartment, which is mainly drying matrix and the inside compartment, which contains cell components with different affinities to water Hence, not all components or functions of cells are equally sensitive to moisture or temperature In other words, there are systems where the inactivation is strongly Figure 2–Relationships between glass transition temperature, water content, and water activity At critical storage temperature a (Tg ), the according storage relative humidity (aw ) and water content of a sample are b and c (adapted and modified from Fonseca and others 2001) Vol 76, Nr 8, 2011 r Journal of Food Science R155 R: Concise Reviews in Food Science Glassy state of dried probiotics R: Concise Reviews in Food Science Glassy state of dried probiotics dependent on temperature and others that are strongly depend on aw (Higl and others 2007) Conclusion and Outlook Despite the current opinion that a glassy state is highly important for the stability of freeze-dried biomaterial and thus has to be maintained throughout the whole production process and storage, no study could empirically confirm its decisive effects, especially in case of probiotics This can be due to the fact that unlike proteins and foods, dried probiotic cells are largely still intact and actually separated from the drying matrices Thus, in case of probiotics, common results relate to the physical state of the surrounding drying matrices regardless of the actual physical state of the cytoplasm For a better understanding of the role of the glassy state on cells, and thus a systematic development of stable freeze-dried probiotics, further studies are encouraged in order to elucidate the physical states of cells during production and storage Further studies with practical implication that should also be addressed are, for example, the role of an annealing step during freezing, optimal moisture content for stable storage of probiotics, changes in probiotic properties due to freeze-drying and storage, and sorption isotherm of dried probiotics References Adams GD, Ramsay JR 1996 Optimizing the lyophilization cycle and the consequences of collapse on the pharmaceutical acceptability of Erwinia L-asparaginase J Pharm Sci 85:1301–5 Andersen AB, Fog-Peterson MS, Larsen H, Skibsted LH 1999 Storage stability of freeze-dried starter cultures (Streptococcus thermophilus) as related to physical state of freezing matrix Food Sci Technol 32:540–7 Buitink J, Leprince O 2004 Glass formation in plant 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storage Proc Biochem 39:1081–6 Zhao G, Zhang G 2005 Effect of protective agents, freezing temperature, rehydration media on viability of malolactic bacteria subjected to freeze-drying J Appl Microbiol 99: 333–8 Optimization of β-Casein Stabilized Nanoemulsions Using Experimental Mixture Design C: Food Chemistry Patrick G Maher, Mark A Fenelon, Yankun Zhou, Md Kamrul Haque, and Yrjăo H Roos Abstract: The objective of this study was to determine the effect of changing viscosity and glass transition temperature in the continuous phase of nanoemulsion systems on subsequent stability Formulations comprising of β-casein (2.5%, 5%, 7.5%, and 10% w/w), lactose (0% to 20% w/w), and trehalose (0% to 20% w/w) were generated from Design of Experiments (DOE) software and tested for glass transition temperature and onset of ice-melting temperature in maximally freeze-concentrated state (Tg & Tm ), and viscosity (μ) Increasing β-casein content resulted in significant (P < 0.0001) increases in viscosity and Tm (P = 0.0003), and significant (P < 0.0001) decreases in Tg A mixture design was used to predict the optimum levels of lactose and trehalose required to attain the minimum and maximum Tg and viscosity in solution at fixed protein contents These mixtures were used to form the continuous phase of β-casein stabilized nanoemulsions (10% w/w sunflower oil) prepared by microfluidization at 70 MPa Nanoemulsions were analyzed for Tg & Tm , as well as viscosity, mean particle size, and stability Increasing levels of β-casein (2.5% to 10% w/w) resulted in a significant (P < 0.0001) increase in viscosity (5 to 156 mPa.s), significant increase in particle size (P = 0.0115) from 186 to 199 nm, and significant decrease (P = 0.0001) in Tg (−45 to −50 ◦ C) Increasing the protein content resulted in a significant (P < 0.0001) increase in nanoemulsion stability A mixture DOE was successfully used to predict glass transition and rheological properties for development of a continuous phase for use in nanoemulsions Keywords: glass transition, nanoemulsion, optimization, stability, viscosity Introduction Food formulations are widely produced by combining carbohydrate, protein, and oil in emulsified systems The characteristics of individual components can be used to control texture and shelf-life (Dickinson and Pawlowsky 1997) Structure through glass formation is provided by carbohydrates Lactose is commonly used for the continuous matrix-forming material during spray drying It is a suitable carbohydrate due to its cheap availability, bland flavor, low viscosity in concentrated solutions, and most importantly its ability to produce a good glass Similarly, trehalose is also a suitable carbohydrate as it has a wide pH-stability range, and is nonreducing in the presence of protein during heat treatment (Higashiyama 2002) Trehalose combined with lactose has been found to delay lactose crystallization while keeping the glass transition temperature of powders (Tg ) constant (Mazzobre and others 2001), due to modifications to the molecular environment by geometric, thermodynamic, or kinetic factors Carbohydrates are often incorporated into dairy protein stabilized nutritional beverages to meet nutritional and structural requirements Typically, these emulsions are stabilized by amphiphilic moieties, such as caseins The caseins are the most abundant proteins (approximately MS 20101303 Submitted 11/17/2010, Accepted 7/7/2011 Authors Maher and Fenelon are with Teagasc Food Research Centre, Moorepark, Cork, Ireland Authors Zhou, Haque, and Roos are with School of Food and Nutritional Sciences, Univ College, Cork, Ireland Direct inquiries to Fenelon (Email: mark.fenelon@teagasc.ie.) C1108 Journal of Food Science r Vol 76, Nr 8, 2011 80% w/w) in bovine milk and adsorb strongly at the oil-water interface during homogenization This layer provides stability to the emulsion during processing and storage and prevents coalescence (Dickinson and Stainsby 1982; Vega and Roos 2006) Caseins also have low heat sensitivity and better surface-active properties compared to whey proteins, which is beneficial for spray drying (Pedersen and others 1998; Hogan and others 2001; Dollo and others 2003; Sliwinski and others 2003) β-casein is the most hydrophobic casein, and steric-stabilizing properties of the protein layer around oil droplets in emulsions are provided by the phosphoserine residues of β-casein (Dickinson 1997a) β-casein is preferentially adsorbed compared to other caseins due to lower interfacial energies, and so β-casein stabilized emulsions are less susceptible to flocculation (Dickinson and others 1987, 1988; Dickinson and Matsumara 1994) Nanoemulsions are emulsions with a particle size in the range of 20 to 200 nm that ensures stability as the diffusion rate, due to Brownian motion, is higher than that for sedimentation and creaming (Solans and others 2005) The glass transition temperature (Tg ) is the temperature at which, upon heating, an amorphous material changes from a glassy, solid-like state to a rubbery, fluid-like state (Langrish and Wang 2006) Glass transition has an effect on many processes and properties encountered in food science In powders, temperatures above the glass transition are found to increase the molecular mobility and free volume, which can result in physico-chemical deterioration, such as crystallization of sugars, stickiness, and caking of powders (White and Cakebread 1966; Slade and Levine 1991) During processing, stickiness may reduce product yields and retard flow of particles leading to operational problems for manufacturing C 2011 Institute of Food Technologists R doi: 10.1111/j.1750-3841.2011.02343.x Further reproduction without permission is prohibited equipment and increased down time (Bhandari and Howes 1999) Also the storage stability and quality changes of food systems are strongly affected by the glass transition (Schenz 1995) Crystallization and caking are time-dependant and are both a function of fluctuation in storage temperature and Tg The higher the temperature above the Tg , the more viscosity decreases and molecular mobility increases leading to crystallization and caking (Bhandari and Howes 1999; Omar and Roos 2007) These phenomena may also cause the continuous matrix to become unstable and release entrapped components, resulting in impaired rehydration properties (Roos and Karel 1991a) It is therefore important that the effects of Tg on food materials, such as emulsions in various states (liquid or dehydrated systems), are taken into account in their processing and storage In this study, an experimental mixture design was generated using Design of Experiments (DOE) software DOE was used to statistically optimize the formulation of nanoemulsions using viscosities and glass transition temperatures at fixed protein con- centrations as response parameters to improve their stability and hence properties as encapsulants Data were analyzed statistically to show by polynomial equations, adjusted R2 and P-values which variables in the formulation have the most significance on selected responses The objective of this study was to produce nanoemulsions with varying viscosities and glass transition temperatures at different protein levels, and observe the effect on mean particle size and stability through the application of a statistical mixture design Materials and Methods D-optimal experimental mixture design The experimental mixture design was set up using the software Design Expert R Software version 7.1.3 (Stat-Ease Inc., Minneapolis, Minn., U.S.A.) A D-optimal design was used with the constraints Lactose + Trehalose + β-casein = 20% w/w with β-casein ≤10% w/w This was used to study the Table 1– Mixture design formulations of carbohydrate-protein mixtures with experimental results of viscosity (μ at 400 s−1 ) and glass transition (Tg , Tm , Tg ) values Run 10 11 12 13 14 15 16 17 Lactose (% w/w) Trehalose (% w/w) β-casein (% w/w) μ (mPa.s) Tg (◦ C) Tm (◦ C) Tg (◦ C) 13.75 10.00 6.25 3.75 20.00 7.50 10.00 10.00 20.00 11.25 15.00 10.00 3.75 20.00 20.00 6.25 13.75 7.50 10.00 10.00 15.00 0.00 10.00 3.75 10.00 0 2.50 10.00 7.50 2.50 5.00 0 5.00 10.00 5.00 10.00 5.00 10.00 3.3 2.1 41.9 2.1 20.1 3.6 2.1 6.9 2.0 2.0 8.3 2.0 78.7 9.4 71.3 3.5 39.3 −43.4 −42.0 −50.1 −43.0 −50.0 −44.7 −42.0 −47.0 −41.1 −41.1 −47.0 −42.0 −49.1 −47.5 −49.0 −46.5 −50.5 −32.7 −32.0 −25.4 −32.0 −28.6 −33.7 −30.0 −31.9 −30.8 −30.5 −30.2 −30.0 −26.0 −27.6 −26.1 −29.4 −25.3 108.5 111.1 119.7 111.0 117.2 111.1 105.7 111.9 109.8 110.0 117.2 104.6 124.0 109.9 123.7 108.8 119.1 Figure 1–Contour and response surface plots of viscosity (at 400 s−1 ) in carbohydrate-protein mixtures comprising of lactose, trehalose, and β-casein (0% to 20% w/w) Vol 76, Nr 8, 2011 r Journal of Food Science C1109 C: Food Chemistry β-casein stabilized nanoemulsions Assessment of daily intake inhabitants of Xiamen, China The SAC/MOHC permits maximum acceptable mercury levels in food of 0.01 to 0.05 mg/kg Cephalopod, Chinese cabbage, eggplant, and lettuce are the group that could be problematic, as they exceed the maximum values permitted for seafood or vegetables Arsenic is a problematic element for humans It is well known that the speciation of As plays an important role in determining As toxicity to humans (Zavala and others 2008), inorganic species (Asi) were thought to be the most toxic in As speciation The results showed that the levels of As in all commodities were between 0.003 mg/kg in apple and 0.10 mg/kg in offal with range of no detectable amount to 0.011 mg/kg and no detectable amount to 1.2 mg/kg, respectively China states 0.05 mg/kg As level for all the foodstuffs analyzed in this study A minority of analyzed vegetables, fruits, and meat samples were found to present higher As contents than the legal limits Table compares the levels of TEs in some vegetables, fruits, and meat from our study with those obtained from studies in other area of China (Song and others 2009), and Egypt (Radwan and Salama 2006), Brazil (Santos and others 2006), and Pakistan (Parveen and others 2003) The values of Cd and Pb were higher in Egypt and Pakistan and lower in Brazil than that found in our similar vegetable and fruit samples The levels of As were of the same magnitude as that of in samples collected from Beijing, China On the other hand, the mean levels of Cd and Pb reported in the present study were comparatively agreed with the level given for similar samples in Spain (Mart´ı-Cid and others 2008) As for the TE levels in cephalopods, concentrations here detected were lower than those found in European marine areas (Storelli 2008) multiplied by the respective consumption rate The EDIs of Cd, Pb, and As were calculated as 0.066, 0.233, and 0.076 μg/kg/d for the mean exposure level, and 0.212, 0.762, and 0.324 μg/kg/d for the 95th percentile exposure level Concerning the EDI of mercury, it has been confirmed that for the majority of seafood, methyl mercury represents the major fraction of total mercury in muscle tissue Therefore, cephalopod is considered separate from other foods in the present analysis The EDI of methyl mercury with that of cephalopod is 0.0062 and 0.01 μg/kg/d, respectively, for the mean and high-end estimate In other food items not related to seafood, total mercury was presumed to be only inorganic mercury The EDIs of total mercury for these food items were 0.014 and 0.051 μg/kg/d, for the middle and upper limits of estimates, respectively The dietary intakes of TEs for inhabitants in Xiamen are similar to that of in other uncontaminated areas in China but significantly lower than that in the other countries (Table 4) For the mean estimate, concerning the foods other than seafood, orange, and pork contribute with approximately 25% and 21%, respectively, to the daily intake of total mercury (Figure 2) The biggest contributions to the daily intake of Pb for inhabitants in Xiamen come from apple and tomato with contributions of 13% and 12%, respectively Tomato, eggplant, and spinach contribute with 15%, 12%, and 11% to the total amount of As ingested via the studied foodstuffs, respectively For the 95th percentile estimate, the contributions of foodstuffs to TE intakes were similar to that of the mean levels (Figure 3) Potential Health Risk of TEs Estimated daily intakes of TEs Potential health risk of individual TEs The EDIs of TEs for inhabitants in Xiamen are listed in Table The RfDs used to calculate the THQ and HI values were 1, To evaluate the EDIs, the mean and the 95th percentile concentra- 0.1, and 0.3 μg kg−1 d−1 for Cd, MeHg, and As according to tion for each TE in each food commodity was calculated, and then the USEPA (IRIS 2007) No oral RfD values were available Table 2–Levels of lead and cadmium in fruits, vegetables, and meats from Xiamen compared with the literature data from similar foodstuffs Toxic element concentrations Beijing, Chinaa,b Present study Foodstuff Chinese cabbage Spinach Legumes Tomato Cabbage Carrot Cauliflower Cucumber Leaf mustard Capsicum Lettuce Eggplant Celery Broccoli Pakchoi cabbage Chicken Pork Offal Apple Grape Peach Orange Pb Cd As 0.029 0.142 0.042 0.066 0.055 0.181 0.046 0.04 0.069 0.034 0.033 0.035 0.072 0.062 0.056 0.008 0.011 0.146 0.078 0.081 0.083 0.083 0.014 0.02 0.005 0.011 0.005 0.011 0.007 0.003 0.007 0.007 0.009 0.01 0.011 0.011 0.01 0.003 0.002 0.055 0.005 0.008 0.004 0.005 0.014 0.033 0.019 0.026 0.013 0.014 0.009 0.017 0.015 0.024 0.014 0.018 0.027 0.026 0.029 0.02 0.023 0.095 0.003 0.003 0.005 0.004 T: Toxicology & Chemical Food Safety a Song and others 2009 b Geometric mean value of c Radwan and Salama 2006 d Parveen and others 2003 e Pb Cd As 0.017 0.018 0.007 0.007 0.005 0.011 0.006 0.017 0.010 0.009 0.014 0.003 0.003 0.022 0.014 0.021 0.015 0.011 0.017 0.007 0.021 0.020 each group of foodstuff in analyzed samples Santos and others 2006 T184 Journal of Food Science r Vol 76, Nr 8, 2011 Egyptc Pb Cd 0.34 0.11 0.26 0.01 0.18 0.01 0.19 Pakistand Pb Cd 1.56 0.33 0.15 1.72 0.36 0.47 0.58 0.21 0.05 0.07 0.02 1.62 0.32 1.3 0.31 0.19 0.87 0.05 0.02 0.76 0.14 0.15 0.04 Brazile Pb Cd 0.047 0.00003 0.00002 0.0004 0.01 0.0075 0.01 0.0006 0.0025 0.0021 0.002 0.0017 0.031 0.004 0.004 0.0003 0.021 0.0001 Assessment of daily intake inhabitants of Xiamen, China for Pb by the USEPA The provisional tolerable weekly intake (PTWI) levels established by the World Health Organization (WHO) were used in place of RfDs for noncarcinogenic risk assessment (FAO/WHO 1993) The PTWI values used was 25 μg/kg/wk for Pb FAO/WHO established a PTWI for inorganic mercury of μg/kg/bw (FAO/WHO 2011), applicable to dietary exposure to total mercury from foods other than seafood in the present study For dietary exposure to mercury from seafood, the RfD value for methyl mercury set by the USEPA was applied The THQs of individual TEs through food consumption for inhabitants in Xiamen are shown in Table The mean and the 95th percentile of the exposure levels were used for calculation of the THQs There are no THQ values over one through consumption of any one kind of foodstuffs listed It can be suggested that health risks associated with TEs exposure for inhabitants of Xiamen were insignificant if they only ingest individual TEs from one kind of foodstuff The total diet THQ of each element (TDHQ, the sum of all types of foodstuffs) is generally less than with the exception of the conservative assessment (the 95th percentile estimate) of As It suggests that inhabitants of Xiamen will not confront with a significant potential health risk by intake of a single element of Cd, Pb, and Hg from foodstuffs However, the TDHQ of As is 1.08 for the 95th percentile exposure level As for methyl mercury, the THQ was 0.062 and 0.1, suggested that health risk from consumption of cephalopod was insignifcant consuming pork is the highest, while celery is the least For the conservative assessment (the 95th percentile exposure level), the biggest contribution to the potential health risk is tomato The HI value expresses the combined noncarcinogenic effects of multiple elements The HI values through consumption for inhabitants in Xiamen are 0.41 and 1.58, based on the mean and the 95th percentile exposure level, respectively This suggests that the high-exposed consumers in Xiamen may experience potential adverse health effects The main relative contributions to the HI are pork (15.5%), tomato (12.3%), cephalopod (8.9%) for the middle limits of estimates, and tomato (12.0%), cabbage (11.7%), and pork (11.6%) for the upper limits of estimates The relative contributions of Cd, Pb, Hg T, and As to the HI are 16.0%, 15.9%, Table –Comparison of dietary intake of toxic elements in Xiamen with previously published results from other parts of the world Cadmium Lead Mercurya Arsenic Origin Xiamen, China Beijing, China 0.066 0.062 0.233 0.014 0.283 Huludao, China 0.749 1.458 0.0382 Tianjin, China 0.14 0.12 Korean 0.26 0.44 Samta, Bangladesh Catalonia, Spain 0.158 1.25 0.029 References 0.076 In this study 0.080 Song and others 2009 Zheng and others 2007 Wang and others 2005 0.7 Lee and others 2006 0.463 Alam and others 2003 3.05 Mart´ı-Cid and others 2008 Rubio and others 2005, 2006, 2008 1.132 Mu˜noz and others 2002 Santos and others 2006 Potential health of combined TEs 0.14 0.85 0.14 Total THQ (TTHQ) of TEs for individual foodstuff is the sum 0.159 1.04 0.095 of the following compositions: TTHQ (individual foodstuff) = Canary Islands, Spain THQ (cadmium) + THQ (lead) + THQ (total mercury) + THQ (arsenic) The potential health risk was calculated by TTHQ of Chile 0.309 3.03 0.0735 different foodstuffs The TTHQ through any individual foodstuff 0.0257 0.4 is all less than one, indicating that there is no significant potential Rio de Janeiro, Brazil health risk for inhabitants in Xiamen by consuming any individual foodstuff For the mean estimate, the potential health risk through a Intake of mercury was calculated with exclusion of cephalopod Table –Estimated dietary intakesa (μg/kg/d) of toxic elements in Xiamen, China (based on the mean and the 95th percentile of exposure levels) Foodstuff Chinese cabbage Spinach Legumes Tomato Cabbage Carrot Cauliflower Cucumber Leaf mustard Capsicum Lettuce Eggplant Celery Broccoli Pakchoi cabbage Chicken Pork Offal Apple Grape Peach Orange Cephalopod Total intake Lead Mercury Arsenic Mean P95 Mean P95 Mean P95 Mean P95 8.52 E–04 3.36 E–03 1.75 E–03 4.62 E–03 2.11 E–03 1.06 E–03 4.01 E–04 4.24 E–04 4.26 E–04 1.09 E–03 3.86 E–04 3.60 E–03 1.97 E–05 6.30 E–04 6.08 E–04 6.44 E–04 1.43 E–03 2.16 E–03 1.87 E–03 4.01 E–04 5.01 E–04 1.55 E–03 3.58 E–02 0.066 2.31 E–03 8.91 E–03 7.36 E–03 1.01 E–02 7.60 E–03 3.18 E–03 1.66 E–03 1.84 E–03 1.22 E–03 2.80 E–03 1.07 E–03 1.11 E–02 4.65 E–05 1.09 E–03 1.89 E–03 4.29 E–03 7.17 E–03 9.72 E–03 7.48 E–03 1.15 E–03 8.77 E–04 6.19 E–03 1.13 E–01 0.212 1.76 E–03 2.39 E–02 1.47 E–02 2.77 E–02 2.32 E–02 1.75 E–02 2.63 E–03 5.65 E–03 4.20 E–03 5.29 E–03 1.42 E–03 1.26 E–02 1.29 E–04 3.55 E–03 3.41 E–03 1.72 E–03 7.89 E–03 5.75 E–03 2.92 E–02 4.06 E–03 1.04 E–02 2.57 E–02 4.03 E–04 0.233 6.33 E–03 7.99 E–02 4.45 E–02 1.27 E–01 1.24 E–01 5.52 E–02 1.41 E–02 2.09 E–02 1.68 E–02 1.71 E–02 4.38 E–03 3.52 E–02 3.09 E–04 9.62 E–03 9.91 E–03 6.44 E–03 3.37 E–02 1.56 E–02 3.74 E–02 5.11 E–03 1.42 E–02 3.28 E–02 5.18 E–02 0.762 1.22 E–04 1.68 E–04 3.51 E–04 8.41 E–04 4.22 E–04 2.90 E–04 5.72 E–05 1.41 E–04 6.08 E–05 3.11 E–04 1.29 E–04 7.19 E–04 5.37 E–06 1.14 E–04 1.82 E–04 1.07 E–03 2.87 E–03 7.87 E–05 7.48 E–04 5.01 E–04 1.25 E–03 3.40 E–03 6.17 E–03 0.014b 3.65 E–04 3.36 E–04 2.10 E–03 2.52 E–03 2.53 E–03 6.76 E–04 3.43 E–04 7.07 E–04 3.04 E–04 9.34 E–04 3.01 E–04 2.16 E–03 1.25 E–05 3.43 E–04 6.08 E–04 4.08 E–03 9.33 E–03 2.36 E–04 1.50 E–03 2.40 E–03 4.88 E–03 1.42 E–02 1.00 E–02 0.051b 8.52 E–04 5.55 E–03 6.66 E–03 1.09 E–02 5.49 E–03 1.35 E–03 5.15 E–04 2.40 E–03 9.12 E–04 3.74 E–03 6.01 E–04 6.47 E–03 4.83 E–05 1.49 E–03 1.76 E–03 4.29 E–03 1.65 E–02 3.74 E–03 1.12 E–03 1.50 E–04 6.26 E–04 1.24 E–03 – 0.076 3.83 E–03 1.70 E–02 3.72 E–02 4.20 E–02 4.18 E–02 5.70 E–03 1.72 E–03 1.67 E–02 4.87 E–03 1.90 E–02 2.79 E–03 3.16 E–02 1.65 E–04 5.15 E–03 6.14 E–03 8.59 E–03 4.59 E–02 2.64 E–02 3.74 E–03 3.51 E–04 1.00 E–03 2.17 E–03 – 0.324 a Estimated b daily intakes were calculated assuming that, when a concentration was below the limit of detection, the concentration was equal to zero Total intake was calculated with exclusion of cephalopod Vol 76, Nr 8, 2011 r Journal of Food Science T185 T: Toxicology & Chemical Food Safety Cadmium Assessment of daily intake inhabitants of Xiamen, China 5.9%, and 62.2% for the mean estimate, and 13.3%, 13.4%, 5.6%, and 67.9% for the 95th percentile estimate Hence, As is the main components contributing to the potential health risk, with total mercury being the least important Estimation of carcinogenic risk of arsenic Concerning the carcinogenic risk of arsenic, the mean R value (mean exposure × slope factor of As) and the 95th percentile R value (95th percentile exposure × slope factor of As) were 1.15×10−4 and 4.86×10−4 , respectively, which are above the ac- ceptable level of in one million (10−6 ) The level of 10−6 was considered to be the generally acceptable carcinogenic risk level in environmental risk management This acceptable level may change in some cases according to environmental policies and may be as high as 10−4 (Sofuoglu and Kavcar 2008) Nevertheless, it raises concern for carcinogenic risk of As in both risk management options However, it should be noted that the RfD of 0.3 μg/kg/d was established for inorganic As compounds, which was considered to be more toxic than organic As The percentage of inorganic As in Figure 2–Individual foodstuff contributions (%) to the total intake of toxic elements (the mean intake estimate) Figure 3–Individual foodstuff contributions (%) to the total intake of toxic elements (the 95th percentile estimate) T: Toxicology & Chemical Food Safety T186 Journal of Food Science r Vol 76, Nr 8, 2011 Assessment of daily intake inhabitants of Xiamen, China Table 5–The target hazard quotient (THQ) values of toxic elements due to foodstuff consumption Cadmium Foodstuff Chinese cabbage Spinach Legumes Tomato Cabbage Carrot Cauliflower Cucumber Leaf mustard Capsicum Lettuce Eggplant Celery Broccoli Pakchoi cabbage Chicken Pork Offal Apple Grape Peach Orange Cephalopod TDHQc Mercurya Lead TTHQb Arsenic Mean P95 Mean P95 Mean P95 Mean P95 Mean P95 8.52 E–04 3.36 E–03 1.75 E–03 4.62 E–03 2.11 E–03 1.06 E–03 4.01 E–04 4.24 E–04 4.26 E–04 1.09 E–03 3.86 E–04 3.60 E–03 1.97 E–05 6.30 E–04 6.08 E–04 6.44 E–04 1.43 E–03 2.16 E–03 1.87 E–03 4.01 E–04 5.01 E–04 1.55 E–03 3.58 E–02 6.57 E–02 2.31 E–03 8.91 E–03 7.36 E–03 1.01 E–02 7.60 E–03 3.18 E–03 1.66 E–03 1.84 E–03 1.22 E–03 2.80 E–03 1.07 E–03 1.11 E–02 4.65 E–05 1.09 E–03 1.89 E–03 4.29 E–03 7.17 E–03 9.72 E–03 7.48 E–03 1.15 E–03 8.77 E–04 6.19 E–03 1.13 E–01 2.12 E–01 4.94 E–04 6.69 E–03 4.12 E–03 7.77 E–03 6.50 E–03 4.90 E–03 7.37 E–04 1.58 E–03 1.18 E–03 1.48 E–03 3.97 E–04 3.52 E–03 3.61 E–05 9.94 E–04 9.54 E–04 4.81 E–04 2.21 E–03 1.61 E–03 8.17 E–03 1.14 E–03 2.91 E–03 7.19 E–03 1.13 E–04 6.52 E–02 1.77 E–03 2.24 E–02 1.25 E–02 3.55 E–02 3.46 E–02 1.54 E–02 3.94 E–03 5.86 E–03 4.72 E–03 4.79 E–03 1.23 E–03 9.87 E–03 8.67 E–05 2.69 E–03 2.78 E–03 1.80 E–03 9.44 E–03 4.37 E–03 1.05 E–02 1.43 E–03 3.96 E–03 9.19 E–03 1.45 E–02 2.13 E–01 2.13 E–04 2.94 E–04 6.14 E–04 1.47 E–03 7.39 E–04 5.07 E–04 1.00 E–04 2.47 E–04 1.06 E–04 5.45 E–04 2.25 E–04 1.26 E–03 9.39 E–06 2.00 E–04 3.19 E–04 1.88 E–03 5.02 E–03 1.38 E–04 1.31 E–03 8.77 E–04 2.19 E–03 5.96 E–03 – 2.42 E–02 6.39 E–04 5.89 E–04 3.68 E–03 4.41 E–03 4.43 E–03 1.18 E–03 6.01 E–04 1.24 E–03 5.32 E–04 1.63 E–03 5.26 E–04 3.78 E–03 2.19 E–05 6.01 E–04 1.06 E–03 7.14 E–03 1.63 E–02 4.13 E–04 2.62 E–03 4.21 E–03 8.55 E–03 2.49 E–02 – 8.91 E–02 2.84 E–03 1.85 E–02 2.22 E–02 3.64 E–02 1.83 E–02 4.51 E–03 1.72 E–03 8.01 E–03 3.04 E–03 1.25 E–02 2.00 E–03 2.16 E–02 1.61 E–04 4.96 E–03 5.88 E–03 1.43 E–02 5.50 E–02 1.25 E–02 3.74 E–03 5.01 E–04 2.09 E–03 4.13 E–03 – 2.55 E–01 1.28 E–02 5.66 E–02 1.24 E–01 1.40 E–01 1.39 E–01 1.90 E–02 5.72 E–03 5.56 E–02 1.62 E–02 6.33 E–02 9.30 E–03 1.05 E–01 5.49 E–04 1.72 E–02 2.05 E–02 2.86 E–02 1.53 E–01 8.80 E–02 1.25 E–02 1.17 E–03 3.34 E–03 7.22 E–03 – 1.08 E + 00 4.40 E–03 2.88 E–02 2.87 E–02 5.03 E–02 2.76 E–02 1.10 E–02 2.96 E–03 1.03 E–02 4.75 E–03 1.56 E–02 3.01 E–03 3.00 E–02 2.26 E–04 6.78 E–03 7.76 E–03 1.73 E–02 6.37 E–02 1.64 E–02 1.51 E–02 2.92 E–03 7.69 E–03 1.88 E–02 3.59 E–02 0.41d 1.75 E–02 8.85 E–02 1.48 E–01 1.90 E–01 1.86 E–01 3.88 E–02 1.19 E–02 6.45 E–02 2.27 E–02 7.25 E–02 1.21 E–02 1.30 E–01 7.04 E–04 2.16 E–02 2.62 E–02 4.18 E–02 1.86 E–01 1.03 E–01 3.31 E–02 7.96 E–03 1.67 E–02 4.75 E–02 1.28 E–01 1.59d a THQ with total mercury intake, so cephalopod was excluded b The total THQs (TTHQ, sum of THQs of each individual element) c TDHQ: the total diet THQ of each element (total diet, sum of all types d of foodstuffs) HI value, sum of THQs values due to consuming the total diet Conclusion The mean concentrations of Cd, Pb, Hg (total and methyl), and As in vegetables, fruits, meat, and seafood were found to be well below the safety concentration limits of China but some samples exceeded their maximum levels Tomato, cephalopod, eggplant, orange, and pork are the main source of TE intakes from foodstuff From the human health point of view, THQ values (1) show that there is no health risk for inhabitants of Xiamen due to intake of individual TEs if only one kind of foodstuff was consumed However, HI for combined TEs due to all the diet was higher than under the 95th percentile scenario, suggesting that highexposed consumers may experience a potential health risk The carcinogenic risk of arsenic in foodstuff is also of concern, since the mean and the 95th percentile carcinogenic rate is above the acceptable risk level of 10−4 References Alam MGM, Snow ET, Tanaka A 2003 Arsenic and heavy metal contamination of vegetables grown in Samta village, Bangladesh Sci Total Environ 308:83–6 Bustamante P, Cosson RP, Gallien I, Caurant F, Miramand P 2002 Cadmium detoxifcation processes in the digestive gland of cephalopods in relation to accumulated cadmium concentrations Mar Environ Res 53:227–41 Cui YJ, Zhu YG, Zhai RH, Huang YZ, Qiu Y, Liang JZ 2005 Exposure to metal mixtures and human health impacts in a contaminated area in Nanning, China Environ Int 31:784–90 Diaz OP, Leyton I, Munoz O, Nunez N, Devesa V, Suner MA, Velez D, Montoro R 2004 Contribution of water, bread, and vegetables (raw and cooked) to dietary intake of inorganic arsenic in a rural village of Northern Chile J Agric Food Chem 52:1773–9 EFSA 2009 Panel on Contaminants in the Food Chain (CONTAM) Scientific Opinion on Arsenic in Food EFSA J 7:1351 198 p Available from: http://www.efsa.europa.eu Accessed May 15, 2010 FAO/WHO 1993 Evaluation of certain food additives and contaminants, 41st report of the Joint FAO/WHO expert Committee on Food Additives FAO/WHO 2011 Summary report of the seventy-second meeting of JECFA Rome Available from: http://www.who.int/ foodsafety/chem/summary72_rev.pdf Accessed Jun 2, 2011 Feig DI, Reid TM, Loeb LA 1994 Reactive oxygen species in tumorigenesis Cancer Res 54:S1890–4 Ge KY 1992 The status of nutrient and meal of Chinese in the 1990s Beijing: People’s Hygiene Press p 415–34 Igwegbe AO, Belhaj H, Hassan TM, Gibali AS 1992 Effect of a highways traffic on the level of lead and cadmium in fruits and vegetables grown along the roadsides J Food Safety 13:7–18 Integrated Risk Information System (IRIS) 2007 US Environmental Protection Agency, Cincinnati, OH Available from: http://www.epa.gov/iris Accessed Aug 26, 2010 Jassir MS, Shaker A, Khaliq MA 2005 Deposition of heavy metals on green leafy vegetables sold on roadsides of Riyadh city, Saudi Arabia Bull Environ Contam Toxicol 75:1020–7 Jorhem L, Sundstroem B 1993 Levels of lead, cadmium, zinc, copper, nickel, chromium, manganese and cobalt in foods on the Swedish market, 1983–1990 J Food Comp Anal 6:223–41 Khan S, Cao Q, Zheng YM, Huang YZ, Zhu YG 2008 Health risks of heavy metals in contaminated soil and food crops irrigated with wastewater in Beijing, China Environ Pollut 152:686–92 Lee HS, Cho YH, Park SO, Kye SH, Kim BH, Hahm TS, Kim M, Ok Lee J, Kim C 2006 Dietary exposure of the Korean arsenic, cadmium, lead and mercury J Food Comp Anal 19: S31–7 Vol 76, Nr 8, 2011 r Journal of Food Science T187 T: Toxicology & Chemical Food Safety food depends on the food type considered Seafood are known to contain organic As The percentage of inorganic As in fish is only up to 11% of total As (EFSA 2009) In contrast, terrestrial foods often have a higher proportion of inorganic arsenic concentrations Cereals and vegetables have been reported to contain on average from 30% to 100% inorganic arsenic (Diaz and others 2004) If we assume an average percentage of 50%, then the mean and 95th percentile intakes of inorganic As in the present study would be 0.038 and 0.162 μg/kg/d, which results in the THQ value of 0.13 and 0.54, respectively Thus, the health risks due to dietary As intake of inhabitants of Xiamen was reduced to a large extent Therefore, a risk assessment not taking into account the different species but taking total As as exclusively inorganic As would lead to a considerable overestimation of the health risk related to dietary As exposure Speciation data are evidently needed to achieve a more accurate and realistic picture Although the daily intake of TEs through food consumption is an important pathway for the dietary exposure, many studies have reported that human beings are also exposed to TEs through drinking water (Mu˜noz and others 2002) As such, exposure estimates are not considered as total dietary exposure to TEs nor we consider residential, or occupational exposures Future research in this area must also take into account the contribution of TEs from other foods and sources in order to make a more realistic risk assessment Assessment of daily intake inhabitants of Xiamen, China Loutfy N, Fuerhacker M, Tundo P, Raccanelli S, El Dien AG, Ahmed MT 2006 Dietary intake of dioxins and dioxin-like PCBs, due to the consumption of dairy products, fish/seafood and meat from Ismailia city, Egypt Sci Total Environ 370:1–8 Mart´ı-Cid R, Llobet JM,Castell V, Domingo JL 2008 Dietary intake of arsenic, cadmium, 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intake in a Spanish Population (Canary Islands) J Agric Food Chem 53:6543–9 Rubio C, Hardisson A, Reguera JI, Revert C, Lafuente MA, Gonz´alez IT 2006 Cadmium dietary intake in the Canary Islands, Spain Environ Res 100:123–9 Rubio C, Guti´errez AJ, Hardisson A, Burgos A 2008 Total dietary intake of mercury in the Canary Islands, Spain Food Addit Contam 8:946–52 SAC/MOHC (The Standardization Administration China/Ministry of Health, China) 2005 Maximum Levels of Contaminants in Foods (GB2762-2005) Beijing Santos EE, Lauri DC, Silveira PCL 2006 Assessment of daily intake of trace elements due to consumption of foodstuffs by adult inhabitants of Rio de Janeiro city Sci Total Environ 327:69–79 Saplakog¢lu U, Iscan M 1997 DNA single-strand breakage in rat lung, liver and kidney after single and combined treatments of nickel and cadmium Mutat Res 394:133–40 T: Toxicology & Chemical Food Safety T188 Journal of Food Science r Vol 76, Nr 8, 2011 Sharma RK, Agrawal M, Marshall FM 2008 Heavy metals (Cu, Cd, Zn and Pb) contamination of vegetables in Urban India: a case Study in Varanasi Environ Pollut 154:254– 63 Sharma RK, Agrawal M, Marshall FM 2009 Heavy metals in vegetables collected from production and market sites of a tropical urban area of India Food Chem Toxicol 47: 583–91 Sofuoglu SC, Kavcar P 2008 An exposure and risk assessment for fuoride and trace metals in black tea J Hazard Mater 158:392–400 Song B, Lei M, Chen T, Zheng Y, Xie Y, Li X, Gao D 2009 Assessing the health risk of heavy metals in vegetables to the general population in Beijing, China J Environ Sci (China) 21:1702–9 Storelli MM 2008 Potential human health risks from metals (Hg, Cd, and Pb) and polychlorinated biphenyls (PCBs) via seafood consumption: estimation of target hazard quotients (THQs) and toxic equivalents (TEQs) Food Chem Toxicol 46:2782–8 USEPA 1986 Guidelines for the health risk assessment of chemical mixtures Federal Register 51:34014–25 USEPA 1999 Guidelines for Carcinogen Risk Assessment, NCEA-F-0644, Review Draft, Risk Assessment Forum Washington, D.C USEPA 2000 Risk-based concentration table Philadelphia, Pa., Washington, D.C Wang XL, Sato T, Xing BS, Tao S 2005 Health risk of heavy metals to the general public in Tianjin, China via consumption of vegetables and fish Sci Total Environ 350:28– 37 Zavala YJ, Gerads R, Găurleyăuk H, Duxbury JM 2008 Arsenic in rice: II Arsenic speciation in USA grain and implications for human health Environ Sci Technol 42:3861–6 Zheng N, Wang QC, Zhang XW, Zheng DM, Zhang ZS, Zhang S 2007 Population health risk due to dietary intake of heavy metals in the industrial area of Huludao City, China Sci Total Environ 387:96–104 Wild Buckwheat Is Unlikely to Pose a Risk to Buckwheat-Allergic Individuals Julie A Nordlee, Rakhi Panda, Joseph L Baumert, Richard E Goodman, and Steve L Taylor Abstract: Buckwheat (Fagopyrum esculentum) is a commonly allergenic food especially in Asia where buckwheat is more commonly consumed Wild buckwheat (Polygonum convolvulus, recently changed to Fallopia convolvulus) is an annual weed prevalent in grain-growing areas of the United States Wild buckwheat is not closely related to edible buckwheat although the seeds have some physical resemblance A large shipment of wheat into Japan was halted by the discovery of the adventitious presence of wild buckwheat seeds over possible concerns for buckwheat-allergic consumers However, IgE-binding was not observed to an extract of wild buckwheat using sera from buckwheat-allergic individuals either by radio-allergosorbent test inhibition or by immunoblotting after protein separation by sodium dodecyl sulfate–polyacrylamide gel electrophoresis Furthermore, the extract of wild buckwheat was not detected in a buckwheat enzyme-linked immunosorbent assay developed with antisera against common buckwheat Thus, wild buckwheat is highly unlikely to pose any risk to buckwheat-allergic individuals The common names of plants should not be a factor in the risk assessment for possible cross-allergenicity Keywords: allergy, buckwheat, grain, wild buckwheat Buckwheat (Fagopyrum esculentum) is a commonly allergenic food in Asian countries probably owing to the frequent consumption of buckwheat noodles (Ebisawa and others 2003) Buckwheat appears on the list of priority allergenic foods in Japan and South Korea (Taylor and Hefle 2005) but not other countries where buckwheat allergy is less frequently encountered Buckwheat allergy can also be quite severe for some affected patients (Noma and others 2001; Moneret-Vautrin and others 2005; Imamura and others 2008) Enzyme-linked immunosorbent assays (ELISAs) for the detection of undeclared buckwheat residues in foods have been developed to support labeling regulations in Japan (Akiyama and others 2004; Panda and others 2010) Wild buckwheat (Polygonum convolvulus; Fallopia convolvulus) is an annual weed prevalent in the midwestern and northern plains of the United States It frequently infests fields of wheat, soybeans, and other grains (Zollinger and others, 2006) It is distantly related to common buckwheat (F esculentum) Wild buckwheat and common buckwheat are in the same genetic family (Polygonaceae) but are distinct at the genus level The use of the name, “buckwheat,” for both of these distantly related plants has the potential to cause confusion for those with buckwheat allergy who must practice avoidance diets and for public health officials attempting to protect such consumers from undeclared buckwheat in foods Although not closely related, these types of seeds have a somewhat similar appearance as both are triangular and dark in color, although wild buckwheat seeds are smaller Recently, the offloading of a large shipment of wheat into Japan was halted by the visual discovery by Japanese inspectors of the ad- MS 20110645 Submitted 5/23/2011, Accepted 7/20/2011 Authors are with Food Allergy Research & Resource Program, Dept of Food Science & Technol., Univ of Nebraska, Lincoln, NE 68583-0919, U.S.A Direct inquiries to author Taylor (E-mail: staylor2@unl.edu) C 2011 Institute of Food Technologists R doi: 10.1111/j.1750-3841.2011.02372.x Further reproduction without permission is prohibited ventitious presence of wild buckwheat seeds mixed with the wheat grain This study was conducted to determine if wild buckwheat would cross-react with buckwheat in the buckwheat ELISA and to determine if wild buckwheat would bind to serum IgE from buckwheat-allergic individuals Materials and Methods Buckwheat, wild buckwheat, and preparation of extracts Buckwheat flour and buckwheat seeds were obtained from a local retail outlet Wild buckwheat seeds were obtained from the grain company whose shipment was embargoed in Japan Buckwheat seeds and wild buckwheat seeds were crushed using dedicated, individual blender jars, and blades Extracts of buckwheat flour, buckwheat seeds, and wild buckwheat seeds were prepared for the ELISA by mixing the flour or crushed grain at a ratio of 1:20 (w/v) with phosphate buffered saline (0.01 M sodium phosphate in 0.85% sodium chloride, pH 7.4) (PBS) + 1% nonfat dry milk (NFDM) with shaking at 60 ◦ C for h in a water bath and were clarified by centrifugation for 30 at 2000 × g A second set of extracts of buckwheat flour, buckwheat seeds, and wild buckwheat seeds were prepared by rocking 1:10 (w/v) with PBS + 0.02% sodium azide, pH 7.4 overnight at ◦ C The extracts were clarified by centrifugation for 30 at 3000 × g The protein concentrations of the extract were determined by the method of Lowry and others (1951) Buckwheat ELISA The buckwheat ELISA was performed as described by Panda and others (2010) using rabbit anti-buckwheat antisera as the capture antibody and goat anti-buckwheat antisera as the detector antibody Human sera Sera were obtained from buckwheat-allergic individuals from a collection of sera from food-allergic subjects maintained by the Vol 76, Nr 8, 2011 r Journal of Food Science T189 T: Toxicology & Chemical Food Safety Introduction Wild buckwheat is unlikely to pose a risk Food Allergy Research and Resource Program at the Univ of Nebraska-Lincoln obtained through an approved Institutional Review Board protocol These sera from North American subjects had specific IgE scores for buckwheat ranging from 12.4 to 43.8 kUA /L (kilo units buckwheat specific IgE per liter serum), a score that is considered as indicative of very strong IgE binding Additionally, the allergic individuals had convincing clinical histories including projectile vomiting, abdominal pain, diarrhea, asthma, swelling of mouth, throat and face, urticaria, and anaphylaxis associated with the ingestion of buckwheat One of these subjects had no other food allergies, while the other two subjects were allergic to both walnut and buckwheat by history and specific IgE results All of these subjects had various inhalant allergies by history including pollen, animal dander, and dust mite allergies RAST inhibition assay A radio-allergosorbent test (RAST) inhibition assay was used to evaluate the ability of the extract of wild buckwheat seeds to compete with buckwheat proteins bound to solid phase for binding of IgE from the pooled sera of buckwheat-allergic individuals using a protocol essentially as described in Hefle and others (1994) Buckwheat proteins from the 1:10 extract of buckwheat flour were bound to a solid phase (cyanogen bromideactivated Sepharose R 4B, Amersham Biosciences Corp., Piscataway, N.J., U.S.A.) which was suspended in RAST-buffer (0.05 M sodium phosphate, 2.5% sodium chloride, 0.2% bovine serum albumin, 0.5% Tween 20, 0.05% sodium azide, pH 7.5) at the rate of 3% (v/v) of swollen solid phase in RAST buffer Serial dilutions of a 1:20 buckwheat flour extract, a 1:20 buckwheat seed extract, and a 1:20 wild buckwheat seed extract were separately mixed with 0.5 mL of a 3% concentration of the suspended buckwheat solid phase and 0.1 mL of a 1:5 dilution of the pooled buckwheat-allergic sera After incubation and removal of unbound human sera by washing, the tubes of solid phase were incubated overnight with antihuman IgE labeled with Iodine 125 (I125) Unbound anti-IgE was removed by washing The amount of IgE bound to the solid phase was determined by measuring the residual radioactivity of the solid phase with a sodium iodide scintillation detector The percent inhibition of IgE binding was calculated with the use of values from buckwheat solid phase samples without inhibitor protein as a measurement of maximal binding were then washed with RAST buffer and then incubated overnight with antihuman IgE labeled with I-125 Blots were washed to remove unbound antihuman IgE, allowed to dry, mounted, and placed between transparencies and exposed to X-ray film for 48 to 96 h at −80 ◦ C Protein bands binding human IgE were visualized after developing the film One blot was not blocked but stained with India ink to confirm protein transfer to the blots India ink staining was achieved by washing the blot × in PBS plus 0.1% Tween 20 and then staining for 15 to 24 h with 0.1% India Ink (Pelikan, Hannover, Germany) in PBS with 0.1% Tween 20 Results and Discussion The extract of wild buckwheat (1:20 w/v) was not detected in the buckwheat ELISA at the lower limit of quantitation of the ELISA of ppm Thus, the IgG antisera used in the buckwheat ELISA was not cross-reactive with any proteins present in the extract of the wild buckwheat seeds Because the RAST inhibition assay evaluates competitive binding to IgE antibodies from the sera of buckwheat-allergic individuals, the results of this assay are much more useful in the assessment of the potential allergenicity of wild buckwheat seeds As shown in Figure 1, the extracts of common buckwheat flour and common buckwheat seeds were able to compete strongly for binding to solid-phase buckwheat proteins By comparison, the extract of wild buckwheat seeds exhibited a very low level of inhibition indicating negligible binding of buckwheat specific IgE to proteins in the wild buckwheat extract On immunoblots (Figure 2), none of the sera from buckwheat-allergic individuals recognized proteins from wild buckwheat that were separated by SDS-PAGE and transferred to the immunoblots In contrast, all sera recognized proteins in the buckwheat flour lanes, although not necessarily the same proteins in each case While research on the identification of buckwheat allergens has been somewhat limited, multiple allergens are known to exist (Nair and Adachi, 1999) and thus the diversity of IgE binding to buckwheat proteins observed with these sera is not surprising No binding was observed with serum from a nonallergic negative control (data not shown) As noted in Fig 2, the degree of protein staining in Lane (wild buckwheat BUCKWHEAT FLOUR RAST-INHIBITION 100 90 80 70 60 50 40 30 20 10 % INHIBITION Electrophoresis and blotting Buckwheat flour slope=33.26 The extracts prepared with PBS + 0.02% sodium azide were separated by sodium dodecyl sulfate–polyacrylamide gel elecBuckwheat seed trophoresis (SDS-PAGE) using 10% to 20% gradient gels Wells slope=32.88 were loaded with 10 μg protein except in the case of the wild buckwheat seed extract that was loaded at μg (maximum amount based on volume of well) A limited quantity of wild buckwheat seeds was available so concentration of the resultant extract was Wild buckwheat seed not possible Electrophoresis was performed according to the manR ufacturer’s instructions for the Mini-Protean II dual slab cell (Bio-Rad Laboratories, Hercules, Calif., U.S.A.) Separated pro1 teins were transferred to polyvinyl difluoride (PVDF) by electroblotting according to the manufacturer’s directions for the Mini LOG NANOGRAM S PROTEIN R Trans-Blot electrophoretic transfer cell (Bio-Rad Laboratories) PVDF blots were blocked with RAST buffer and then incubated Figure 1–Comparison of wild buckwheat with common buckwheat and overnight either with control serum or serum samples from the buckwheat flour to inhibit IgE-binding from human sera of buckwheatbuckwheat-allergic subjects diluted 1:10 in RAST buffer Blots allergic individuals as shown by RAST inhibition T: Toxicology & Chemical Food Safety T190 Journal of Food Science r Vol 76, Nr 8, 2011 Wild buckwheat is unlikely to pose a risk Based upon these results, wild buckwheat (P convolvulus) is highly unlikely to present a risk to buckwheat-allergic individuals The profound difference in IgE binding between common buckwheat (F esculentum) and wild buckwheat is sufficient to conclude that additional evidence of the lack of allergenicity of wild buckwheat is not needed No evidence of the allergenicity of wild buckwheat exists in the published clinical literature despite the high likelihood that some intake of wild buckwheat seeds is likely to have occurred due to grain contamination No reason exists to suggest that wild buckwheat seeds might pose a risk to buckwheatallergic individuals Wild buckwheat is not closely related to edible buckwheat even though the seeds of wild buckwheat bear some physical resemblance of edible buckwheat seeds The common names of plants should not be a factor in the risk assessment for possible cross-allergenicity Instead, the botanical relationships are more likely to predict potential risk References extract) obtained with India ink is less than anticipated by the comparative amounts of protein loading of the wells in Lanes and (10 compared with μg) This may reflect the comparative ability of India ink to bind to the proteins from the different extracts or to the possible existence of a greater diversity of protein in the wild buckwheat extract Vol 76, Nr 8, 2011 r Journal of Food Science T191 T: Toxicology & Chemical Food Safety Figure 2–SDS-polyacrylamide gel electrophoresis of molecular weight markers (Lane 1), buckwheat flour (Lane 2), buckwheat seed extract (Lane 3), and wild buckwheat seed extract (Lane 4) Protein stain is shown on the leftmost panel while immunoblotting with sera from different buckwheat-allergic subjects is shown on the following panels Akiyama H, Nakamura Y, Harikai N, Watanabe H, Iijima K, Yamakawa H, Mizuguchi Y, Yoshikawa R, Yamamoto M, Sato H, Watai M, Arakawa F, Ogasawara T, Nishihara R, Kato H, Yamauchi A, Takahata Y, Morimatsu F, Mamegoshi S, Honjoh T, Watanabe T, Sakata K, Imamura T, Toyoda M, Matsuda R, Maitani T 2004 Inter-laboratory evaluation of studies for establishment of notified ELISA methods for allergenic substances (buckwheat) J Food Hyg Soc Japan 45:313–8 Ebisawa M, Ikematsu K, Imai T 2003 Food allergy in Japan Allergy Clin Immunol Int 15:214–7 Hefle SL, Lemanske RF Jr Bush RK 1994 Adverse reaction to lupine-fortified pasta J Allergy Clin Immunol 94:167–72 Imamura T, Kanagawa Y, Ebisawa M 2008 A survey of patients with self-reported severe food allergies in Japan Pediatr Allergy Immunol 19:270–4 Lowry OH, Rosebrough NS, Fair AL, Randall RJ 1951 Protein measurement with the Folin phenol reagent J Biol Chem 93:265–75 Moneret-Vautrin DA, Morisset M, Flabbee J, Beaudouin E, Kanny G 2005 Epidemiology of life-threatening and lethal anaphylaxis: a review Allergy 60:443–51 Nair A, Adachi T 1999 Immunodetection and characterization of allergenic proteins in common buckwheat (Fagopyrum esculentum) Plant Biotechnol 16:219–24 Noma T, Yoshizawa I, Ogawa N, Ito M, Aoki K, Kawano Y 2001 Fatal buckwheat dependent exercise-induced anaphylaxis Asia Pacific J Allergy Immunol 19:283–6 Panda R, Taylor SL, Goodman RE 2010 Development of a sandwich enzyme-linked immunosorbent assay (ELISA) for detection of buckwheat residues in food J Food Sci 75:T110–7 Taylor SL, Hefle SL 2005 Food allergies and intolerances In: Shils ME, Shike M, Ross AC, Caballero B, Cousins RJ, editors Modern nutrition in health and disease 10th ed Philadelphia, Pa.: Lippincott Williams & Wilkins p 1512–30 Zollinger R, Peterson D, Moechnig M 2006 Biology and management of wild buckwheat: the glyphosate, weeds, and crops series GWC-10 Purdue Extension Available from: www.ces.purdue.edu Effects of Raspberry Phytochemical Extract on Cell Proliferation, Apoptosis, and Serum Proteomics in a Rat Model Hong-Sheng Chen, Ming Liu, Li-Jun Shi, Jin-Lu Zhao, Chun-Peng Zhang, Luo-Qiang Lin, Yan Liu, Shu-Jun Zhang, Jun-Chao Jin, Lei Wang, Bao-Zhong Shen, and Jia-Ren Liu The red raspberry extract possesses potent antioxidant capacity and anticancerous activity in vitro and in vivo The objective of this study was to determine whether red raspberry extract affected the cell cycle, angiogenesis, and apoptosis in hepatic lesion tissues from a rat model induced by diethylnitrosamine (DEN) as well as changes of serum proteomics Rats were treated with red raspberry extract (0.75, 1.5, or 3.0 g/kg of body weight) by gavage starting h after DEN administration and continued for 20 wk Red raspberry extract inhibited cell proliferation, vascular endothelial growth factor VEGF expression, and induced apoptosis in the hepatic lesion tissues In addition, protein peaks (2597.93 and 4513.88 m/z) were identified to differentially express in the 3.0 g/kg body weight and positive control groups by serum proteomics These results suggest that a dietary supplement with red raspberry effectively protects against chemically induced hepatic lesions in rats Abstract: Keywords: apoptosis, hepatic lesions, proliferating cell nuclear antigen, red raspberry extract Introduction Primary liver cancer, commonly known as hepatocellular carcinoma (HCC), is the 5th most frequent cause of death in males and is the 8th most frequent cause of death in females throughout the world Liver cancer is the 3rd leading cause of death worldwide with an estimated 680000 deaths occurred in 2007 (American Cancer Society, Atlanta, Ga., U.S.A 2007) Carcinogenesis proceeds in steps: initiation, promotion, and progression It has been reported that more than 30% of human cancers could be prevented by one strategy of appropriate dietary modification (Doll and Peto 1981; Willett 2002) Epidemiological and laboratory studies showing a protective effect of diets rich in fruits and vegetables against cancer have focused attention on the possibility that biologically active phytochemicals exert anticarcinogenic activity (Block and others 1992; Liu and others 2008, 2009a, 2009b, 2010a, 2010c) Red raspberries are one of very significant parts of the diet and are also an important source of antioxidant phenolic compounds in the western world Red raspberries are a delicious fruit that can be consumed fresh or in the form of products such as jams, juices, and liquors Phenolic compounds in the extract of red raspberry were shown to have a strong antioxidant capacity MS 20110719 Submitted 6/9/2011, Accepted 7/20/2011 Authors H.-S Chen, M Liu, J.-L Zhao, C.-P Zhang, L.-Q Lin, Y Liu, S.-J Zhang, J.-C.Jin, and L Wang are with Treatment Center of Oncology, the Fourth Affiliated Hospital of Harbin Medical Univ., 37 YiYuan Street, NanGang District, Harbin 150001, P.R China Author L.-J Shi is with the First Affiliated Hospital of Harbin Medical Univ., 23 You Zheng St., NanGang District, Harbin 150001, P.R China Author B.-Z Shen is with Key Laboratory of Molecular Imaging in Heilongjiang, 150001, P.R China Author J.-R Liu is with Public Health College, Harbin Medical Univ., 157 BaoJian Rd., NanGang District, Harbin, 150081, P.R China Author J.-R Liu is currently with Harvard Medical School, 300 Longwood Ave., Boston, MA 02115, U.S.A Direct inquiries to author J.-R Liu (E-mail: Jia-ren.liu@childrens.harvard.edu) T: Toxicology & Chemical Food Safety Authors H.-S Chen, M Liu, and L.-S Shi have equally contributed to this work T192 Journal of Food Science r Vol 76, Nr 8, 2011 (Wang and Lin 2000; Parry and others 2005) and anticancer activities (Seeram and others 2006; Boivin and others 2007; Aiyer and others 2008a) In previous studies, raspberry extract and its constituents had an antioxidant capacity and inhibited the growth of mammary, oral, colon, prostate, and liver cancer cells in vitro in a dose-dependent manner (Liu and others 2002; Seeram and others 2006) and induced apoptosis of MCF-7 breast cancer cells (Seeram and others 2006) In our previous study (Liu and others 2010d), fresh red raspberry extract at doses of 0.75, 1.5, or 3.0 g/kg body weight significantly inhibited hepatic lesions in a diethylnitrosamine (DEN)-induced rat model and demonstrated a dosedependent manner To further determine the possible mechanism of hepatic lesion inhibition by raspberry, the following study was performed The objective of the present study was to determine whether raspberry phytochemical extract affected (1) serological proteomics; (2) cell proliferation; and (3) cell apoptosis in hepatic tissues from a model of rat induced by DEN Methods Red raspberry extraction The extraction of red raspberry (Rubus idaeus L.) and control was described in our previous study (Liu and others 2009a, 2010d) The red raspberries (Rubus idaeus L.) were purchased from a supermarket in Northeast of China Briefly, 100 g of fresh red raspberries were weighed and homogenized with chilled 80% acetone (1:2, w/v) using a chilled Waring blender for The sample was then further homogenized using a Polytron homogenizer for an additional The homogenates were filtered through Whatman #1 filter paper on a Buchner funnel under vacuum The filtrate was evaporated at 45 ◦ C until approximately 90% of the filtrate had been evaporated The raspberry extract was standardized to contain 437.62 ± 29.94 mg total phenolics (gallic acid equivalents) per 100 g raspberries The standardized raspberry extracts were frozen and stored at −80 ◦ C until use in the feeding C 2011 Institute of Food Technologists R doi: 10.1111/j.1750-3841.2011.02373.x Further reproduction without permission is prohibited study Control extract was also prepared using the same extraction assay (Promega Corp., Madison, Wis., U.S.A.) (Liu and others 2008, 2009a) The slices of hepatic lesion tissues were deparafsolvents and procedures without red raspberries finized in xylene and rehydrated through graded alcohol, then Animal treatment and experimental design washed in 2% NaCl in phosphate-buffered saline buffer, fixed in Pathogen-free male Wistar rats, 130 to 140 g, were purchased 4% paraformaldehyde, treated with proteinase K (20 μg/mL) for from Sino-British SIPPR/BK Lab Animal Ltd (Shanghai, China) 30 min, refixed with 4% paraformaldehyde, and incubated with The rats adapted immediately to the AIN-93M diet and were terminal deoxynucleotidyl transferase (TdT) reaction mix in a huhoused in a room with a 12-h light/12-h dark cycle The rats midified box for h at 37 ◦ C The slices were washed in 2× SSC were acclimated to the surroundings in the animal room for (0.15 mol/L sodium chloride and 0.015 mol/L trisodium citrate, wk prior to the initiation of the experiment Rats were handled saline-sodium citrate [SSC] buffer) for 15 and then blocked according the Guide for the Care and Use of Laboratory Animals in 3% H2 O2 for The slides were then incubated with National Research Council (1985) The rats were randomly as- streptavidin−horseradish peroxidase and colorized with DAB All signed to groups (n = 20/group) Three raspberry treated and sections were counterstained with hematoxylin Controls were positive control groups of rats were given 10 mg/kg body weight subjected to the same protocol with the omission of the TdT reof DEN (Sigma Chemical Co., St Louis, Mo., U.S.A.) water so- action mix Microscopic images were measured at a magnification lution by gavage once and continue to drink 0.025% DEN water of 400× Two thousand cells were counted in visual fields ranad libitum for 20 wk; the 5th group which received no DEN but domly in each section, and an average of positive number in each was given mL of distilled water served as the negative control group was examined to determine the number of apoptotic cells, group Rats were administered the control or red raspberry extract which were identified by a brown stain over the nuclei (mean ± starting h after DEN administration and continuing for 20 wk S.D.) Three levels of low, middle, and high doses of red raspberry extract were given to the rats by gavage corresponding to 0.75, 1.5, or Serological proteomics analysis 3.0 g fresh raspberry/kg/day body weight raspberry extract, reThe different protein analysis of rat serum was described in spectively The control groups fed with control extraction Animals our previous study (Liu and others 2010b) Briefly, the serum were weighed weekly All survivors were killed under anesthesia samples from experimental or controls groups were centrifuged (ketamine 5.0 mg/kg body weight) at the end of the 20th wk af- at 10000 rpm for at ◦ C Taking 10 μL of the serum ter DEN administration and arterial blood samples were collected sample, it was filled with 20 μL of U balanced solutions (9 from each rat The sera from these rats were extracted for analysis mol Ureas, 2% CHAPSs, 50 mmol/L Tris-HCl, pH 9.0, and 1% of serological proteomics DL-dithiothreitol) into the bores with shaking The samples were shaken in an ice bath (MS1 Minishaker) at a rate of 400 to 600 Immunohistochemical analysis of proliferating cell nuclear rpm for 30 and then 360 μL of natrium aceticum buffer was antigen and vascular endothelial growth factor expression put (50 mmol/L NaAc, pH 4.0) into 30 μL of dilution solution in hepatic lesion tissue with shaking The chip of the WCX2 (Ciphergen Co., Fremont, The sections of hepatic lesion tissues were deparaffinized in xy- Calif., U.S.A.) was placed into the bioprocessor, and after filling lene and rehydrated through graded alcohol (100%, 95%, 70%, each bore with 200 μL of natrium acetic buffer and spinning the and 50%) to distill water The sections were incubated for 10 bioprocessor at a rate of 400 to 600 rpm for min, the buffer at 95 to 100 ◦ C in 10 mmol/L sodium citrate buffer (pH 6.0) En- was removed The same process mentioned above was repeated dogenous peroxidases were inactivated by immersing the sections Each bore of the bioprocessor was filled with 100 μL of the in hydrogen peroxide for 10 min, and then the sections were incu- sample, agitated at a rate of 400 to 600 rpm for h at ◦ C bated for 10 with 10% normal goat serum to block nonspecific (iced bath) After removing the sample, 200 μL sodium acetate binding The sections were subsequently incubated overnight at buffer (50 mmol/L NaAc, pH 4.0, or the binding buffer in kit) ◦ C with antiproliferating cell nuclear antigen (PCNA) antibody was added to each bore and was spun at a rate of 400 to 600 (monoclonal mouse, F-2, IgG 2a, 1:100 dilution) or anti-vascular rpm for at room temperature This process was repeated endothelial growth factor (VEGF) antibody (polyclonal rabbit, again Subsequently, 200 μL of HPLC flow phase was added to 1:20 dilution) (Santa Cruz Biotechnology, Inc., Santa Cruz, Calif., each bore, and then discarded immediately This procedure was U.S.A.) The sections were then incubated with biotinylated or repeated twice The chip was taken out and 0.5 μL of sinapinic antirabbit/antimouse IgG (ZYMED, Lab, Inc., Carlsbad, Calif., acid (SPA; 50% CANs [trifluoroacetic acid : acetonitrile + : U.S.A.) for 30 min, followed by peroxidase-conjugated strepta- 1] + 0.5% trifluoroacetic acid) solution was added to each well vidin (ZYMED, Lab, Inc.) for 30 The chromogenic reaction after exsiccation After sample exsiccation, SPA was added again was developed with 3,3 -diaminobenzidine tetrachloride (DAB) The dried samples were analyzed by the surface-enhanced laser for min, and all sections were counterstained with hematoxylin desorption/ionization–time of flight mass spectrometry (SELDIThe same protocol was applied to the controls with the omission TOF-MS) system of the primary antibody Microscopic images were measured at a Chips were placed in the SELDI-TOF-MS system (Ciphergen magnification of 400× Two thousand cells in each section were Company), and time-of-flight spectra were generated by averaging counted in visual fields, and an average of over to tissue masses 192 laser shots collected in the positive mode at laser intensity in each group were analyzed to determine if the cells positively scale 215 and detector sensitivity scale The mass range from stained for specific protein expression (mean ± S.D.) molecular weight 10000 to 20000 Da or the highest 50000 Da was selected for analysis because this range contained the majority Determination of apoptosis by TUNEL assay of the resolved protein/peptides The range of data collection was The effect of red raspberry extract on apoptosis in rat hep- designed from 10000 to 50000 m/z (mass-to-charge) The data were analyzed by Ciphergen ProteinChip software atic lesion tissues was evaluated by terminal deoxynucleotidyl transferase-mediated deoxyuridine nick end labeling (TUNEL) 5.1 (Ciphergen Company) The protein mass peak which had Vol 76, Nr 8, 2011 r Journal of Food Science T193 T: Toxicology & Chemical Food Safety Raspberry phytochemical extract inhibits hepatic lesions of rat Raspberry phytochemical extract inhibits hepatic lesions of rat Each serum sample was processed at least in triplicate to confirm reproducibility in resolving the proteins One hundred and two mass peaks of protein were acquired from 2000 to 20000 peaks of m/z Two m/z peaks of protein were found by BiomarkerWizard Software’s analysis (3.1) when compared to the control groups We identified 2597.93 and 4513.88 m/z peaks to differentially express in the high dose of red raspberry treatment and positive control group (Table and Figure 4) There was 3.3 times higher of 2597.93 m/z peak in the high dose of red raspberry group Statistical analysis Data were expressed as mean ± S.D The expression of PCNA, than that in the positive control group (P = 0.007) and 1.75 times VEGF, and TUNEL in hepatic lesion tissues was analyzed us- higher of 4513.88 m/z peak than that in the positive control group ing Student’s t-test, Welch’s t-test, or analysis of variance Data (P = 0.021) analyses were generated and plots were constructed using SPSS for Windows Version 19.0 (SPSS Inc., Chicago, Ill., U.S.A.) and Discussion Red raspberries contain a variety of phenolic compounds SigmaPlot Version 11.2 for Windows (Systat Software Inc., San Jose, Calif., U.S.A.) Statistical significance was set at P < 0.05, such as flavonoids, phenolic acids, and tannins and are a rich source of anthocyanins, flavonols, and ellagitannins, and also conand all P-values were unadjusted for multiple comparisons tribute to the dietary intake of these phenolics (Hakkinen and others 1999; Maatta-Riihinen and others 2004) In a previously Results study, red raspberry extract significantly inhibited the proliferaImmunohistochemistry for PCNA and VEGF expression tion of HepG2 human liver cancer and CaCo-2 human colon To determine whether red raspberry extract affected cell pro- cancer cells in a dose-dependent manner (Liu and others 2002; liferation and angiogenesis of hepatic lesion tissues, PCNA and McDougall and others 2008) The major groups of phenolic VEGF were determined in the rat hepatic tissues by immunohis- compounds present in red raspberries are reported to be anthotochemical skills PCNA is a common index for the proliferation cyanins, flavonols, flavanols, ellagitannins, gallotannins, proanthoof hepatic tumor cells at the early and late G1 stage As shown in cyanidins, and phenolic acids (Wu and Prior 2005) as well as Figure 1, the expression of PCNA was the lowest in the negative ellagic and gallic acid The major anthocyanins and tannins identicontrol group The expression of PCNA in hepatic tumor tissues fied are as follows: cyanidin-3-sophoroside, cyanidin-3-glucoside, of red raspberry-treated groups (237 ± 39, 178 ± 15, and 63 ± pelargonidin-3-glucoside, and predominantly hydrolyzable tan25 per 500 cells in low-, middle-, and high-dose groups, re- nins (ellagitannins and gallotannins) The mixture of 15 anthospectively) significantly decreased in comparison with the control cyanidin 3-O-glycosides, cyaniding 3-glucoside, ellagic acid, and group (271 ± 39 per 500 cells) (P < 0.05 or P < 0.01) (Figure 1) gallic acid inhibited cell proliferation of HT-29 in a concentrationCompared with the positive control group, the inhibitory rates of dependent manner (Wu and others 2007) In addition, red PCNA expression were 13%, 34%, and 77% in the low-, middle-, raspberry extract and its components also reduced endogenous and high-dose groups, respectively, demonstrating a clear dose re- oxidative DNA damage in vitro and in vivo (Aiyer and others sponse 2008b) In our previous study (Liu and others 2010d), rats fed As shown in Figure 2, the expression of VEGF was also the levels of 0.75 (low), 1.5 (medium), and 3.0 (high) g fresh red lowest in the negative control group VEGF expression in the raspberries/kg body weight per day starting h after DEN adred raspberry-treated groups was significantly lower than that in ministration and continuing for 20 wk until the end of the exthe positive control group (181 ± 26 per 500 cells) (P < 0.05 periment The positive control group with carcinogen DEN deor P < 0.01) (Figure 3) Compared with the positive control veloped hepatic nodule formation with 45.0% during a 20-wk group, the inhibitory rates of VEGF expression were 45%, 62%, study and no nodules were detected in the negative control group and 88% in the low-, middle-, and high-dose groups, respectively, without DEN A dose-dependent inhibition of nodule formation demonstrating a clear dose-dependent manner by fresh red raspberry extract was observed Pathological findings also showed that there was hepatocellular adenocarcinoma in Apoptosis induction in hepatic lesion tissue the control group and 25.0%, 40.0%, and 0% in the levels of Apoptosis was detected in hepatic lesion tissues using the low, medium, and high doses of red raspberry extract, respectively, TUNEL assay As shown in Figure 3, a low rate of apoptosis was during the 20-wk study (P < 0.05) The ultrastructural findings observed in the hepatic tissues from the negative control group also showed that red raspberry diet could reduce markers of the (3 ± per 500 cells) Occurrences of apoptosis in hepatic tis- diagnostic features of the neoplasm In addition, at the terminasues in the middle and high doses of red raspberry treatments tion of the experiment (20 wk) final body weight and the relative were significantly higher than those in the control groups (P < weights of liver were not significantly different between the nega0.05 or P < 0.01) Apoptosis expression was increased by 1.6 and tive control and raspberry treated groups (Liu and others 2010d) 3.6 times in the middle- and high-dose groups, respectively, com- It indicates that there was no toxicity observed in the animals fed pared to the positive control group (27 ± 11 per 500 cells) No the red raspberry extract at the doses tested In our study, we significant differences were found between the low and positive not analyze the phytochemical composition of this extraction as control groups Apoptosis was induced by red raspberry extract in well as the bioavailability of some compounds This work will be a dose-dependent manner formed in the future study PCNA is a nuclear protein which functions as an auxiliary Proteomic analysis of serum protein for DNA polymerase δ and is an absolute requirement The weak cation exchanger (WCX)-type chip was used for DNA synthesis PCNA, a key protein in DNA replication throughout this study because of the presence of differential peaks and DNA damage repair, is a stable cell-cycle regulated nuclear the different expressions was found, and the data were imported with the Excel format into Ciphergen ProteinChip Software 5.1 Then, all the protein mass peaks which had significantly different expression were listed (P < 0.05), the best alignment combination was selected, and results were analyzed by Biomarker Wizard 3.1 Each serum sample was performed at least in triplicate to confirm reproducibility and reduce bias T: Toxicology & Chemical Food Safety T194 Journal of Food Science r Vol 76, Nr 8, 2011 Raspberry phytochemical extract inhibits hepatic lesions of rat protein that is expressed during the cell cycle and whose rate of synthesis is correlated directly with the proliferative rate of cells (Bravo and others 1987) The higher specificity of PCNA for the S-phase might be thought to be advantageous over other proliferation markers as it should label those cells that have passed the important G1/S boundary of the cell cycle PCNA has been shown to be more sensitive in detecting proliferating cells in some tumors (Fairman 1990; Hall and Levison 1990; McCormick and Hall 1992) VEGF is an endothelial cell mitogen that initiates and promotes neovascularization and endothelial cell proliferation, and Figure 2–Effects of different doses of fresh red raspberry extract on expression of VEGF in the hepatic lesion tissues in vivo High-power view (400×) of VEGF expression: (A) positive control group (control +); (B) negative control group (control –); (C) low; (D) middle; and (E) high doses of red raspberry extract The expression of VEGF was visualized with DAB; positive cells were detected as the brown color stained in the cytoplasma; (F) bars with no letters in common are significantly different (P < 0.05 or P < 0.01) Vol 76, Nr 8, 2011 r Journal of Food Science T195 T: Toxicology & Chemical Food Safety Figure 1–Effects of different doses of fresh red raspberry extract on expression of PCNA in the hepatic lesion tissues in vivo High-power view (400×) of PCNA expression: (A) positive control group (control +); (B) negative control group (control –); (C) low; (D) middle; and (E) high doses of red raspberry extract The expressions of PCNA were visualized with DAB; positive cells are reddish-brown in color; (F) bars with no letters in common are significantly different (P < 0.05 or P < 0.01) Raspberry phytochemical extract inhibits hepatic lesions of rat it was initially identified as a vascular permeability factor VEGF has a major effect in regulating angiogenesis, and its expression has been shown to correlate with carcinogenesis (Grizzi and others 2007) VEGF is also one of the most attractive targets for molecular therapy against HCC (Midorikawa and others 2010) In this study, very low levels of PCNA and VEGF expressions were observed in the negative control group, and both were highly expressed in the positive control group Red raspberry extract significantly decreased both of PCNA and VEGF expressions in a dose-dependent manner This indicates that PCNA and VEGF expressions are the sensitive marker of cell proliferation and angiogenesis of hepatic lesion tissues The red raspberry extract reduced both of markers in a dose-dependent manner, indicating that the extract may inhibit cell proliferation and angiogenesis in the hepatic lesion tissues of DEN-treated rats In contrast to nonspecific cellular necrosis, apoptosis is characterized by a specific pattern of DNA degradation that exposes -OH ends As a result, apoptotic cells within tissue sections were identified by the TUNEL of exposed -OH ends with nonisotopically digoxigenin-labeled dUTP (Gavrieli and others 1992; Huerta and others 2007) The TUNEL assay is widely used and accepted as a tool for indicating apoptosis In our study, red raspberry extract induced apoptosis in hepatic tumor tissues from the red raspberry-treated groups in a dose response Thus, apoptosis induction may be one of mechanisms in the preventive hepatic lesions by red raspberry In our study, red raspberry extract not only inhibited cell proliferation and angiogenesis, but also initiated apoptosis in hepatic lesion tissues of DEN-treated rats In addition, there were differential proteins or peptides between the high and positive control groups We also need to further determine the relationship be- tween differential proteins or peptides and cell proliferation or angiogenesis or apoptosis in the promotion and progression of hepatocarcinogenesis Serum proteomics studies were traditionally based on 2-dimensional gel electrophoresis, although this method is insufficiently sensitive to detect low-abundance proteins (Chignard and Beretta 2004) However, SELDI-TOF-MS has been advocated as a superior method to identify unique serum protein fragments (Poon and others 2003) The m/z ratio of these peptide fragments is then determined, which generates a peptide mass fingerprint Two sets of two distinct SELDI peaks were identified to allow differentiation between Chinese HCC patients and normal subjects or cirrhosis patients, respectively (Wang and others 2005) In our previous study (Liu and others 2010b), 100 serum samples from 52 cases of colorectal cancer, 27 cases of colorectal benign disease, and 21 cases of healthy controls were examined by SELDI-TOFMS with WCX2 protein chips and diagnostic models (I, II, and III) were set up These models were combined with protein mass peaks to discriminate colorectal cancer, colorectal benign diseases, and healthy controls There were over 80% accuracy, sen- Table 1–Serum proteomics studies showed that peaks of protein were identified to differential express in high and DEN groups based on a SELDI-TOF method Peaks of protein (m/z) 2597.93 4513.88 High group DEN group 11.9203 ± 2.063∗∗ 7.564 ± 1.522∗ 3.6141 ± 2.368 4.334 ± 0.904 ∗∗ P < 0.01, ∗ P < 0.05, compared to the DEN group DEN = diethylnitrosamine; high group: DEN + 3.0 g red raspberry/body weight T: Toxicology & Chemical Food Safety Figure 3–Effects of different doses of fresh red raspberry extract on apoptosis in the hepatic lesions tissues in vivo High-power view (400×) of apoptosis expression: (A) positive control group (control +); (B) negative control group (control –); (C) low; (D) middle; and (E) high doses of red raspberry extract Apoptotic cells were observed by TUNEL assay The apoptotic cells in hepatic tumor tissues were observed as the brown color stained in the nucleus (F) Bars with no letters in common are significantly different (P < 0.05 or P < 0.01) T196 Journal of Food Science r Vol 76, Nr 8, 2011 Raspberry phytochemical extract inhibits hepatic lesions of rat Figure 4–Protein profiling on WCX2 chips Representative overview of protein profiling on WCX2 chips show spectral map (left panel) and gel view (right panel) of the serum samples SELDI analysis of rat serum for proteomic pattern in the high dose of red raspberry treatment (H), negative control (C), and positive control (D) samples with mass spectra (left) and gel view (right) Differential expressed proteins were found in m/z values of (A) 2597.93 Da and (B) 4513.88 Da Conclusion In summary, our data indicate that fresh red raspberry extract potently inhibited cell proliferation, angiogenesis, and induce apoptosis in hepatic lesion tissues of DEN-treated rats Two different new proteins or peptides were found between the high and positive control groups These results suggest that a dietary intervention effectively protects against chemically induced hepatic lesions in this model Thus, red raspberry may be a potential source of chemical compounds that have an antiproliferation effect on cancer cells and its exact mechanism(s) warrants further study Acknowledgments This work was supported by the Specialized Research Fund for the Doctoral Program of Higher Education, People’s Republic of China (nr 20092307110019), the Natural Science Foundation of Heilongjiang Province, People’s Republic of China (nr LC201009), and Technological Innovation Project of Harbin Vol 76, Nr 8, 2011 r Journal of Food Science T197 T: Toxicology & Chemical Food Safety sitivity, and the particularity of cross verification of these models for screening patients The SELDI-TOF has been used by several groups to screen for potential biomarkers of ovarian, prostate, breast, bladder, pancreatic, and many other cancers (Xiao and others 2005) In this study, sera from rats in the raspberry-treated and controls groups were analyzed by SELDL-TOF The 2597.93 and 4513.88 m/z peaks were identified to differentially express in the high dose of red raspberry and positive control group These may be new targets of prevention and therapy in hepatic lesions However, we did not analyze these protein peaks in other red raspberry-treated groups because of inadequate sera We also did not get the amino acid sequence of these protein peaks Thus, we further need to determine which kinds of proteins are these and their effects on the promotion and progression of hepatocarcinogenesis We will analyze structures and functions of these proteins and further determine whether the same or similar proteins also exist in human patients as well as the relationship between proteins and human hepatic tumors Raspberry phytochemical extract inhibits hepatic lesions of rat Science and Technology Bureau, People’s Republic of China (nr 2009RFLXS002) Hong-Sheng Chen, Ming Liu, and Li-Jun Shi are co-first authors Jia-Ren Liu, Ming Liu, and Bao-Zhong Shen are co-corresponding authors The authors declare that they have no competing interests References Aiyer HS, Kichambare S, Gupta RC 2008a Prevention of oxidative DNA damage by bioactive berry 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Journal of Food Science C1125 C: Food Chemistry Discrimination of Chinese Vinegars Based on Headspace Solid-Phase Microextraction-Gas Chromatography Mass Spectrometry of Volatile Compounds and Multivariate. .. Chemistry Discrimination of Chinese vinegars Discrimination of Chinese vinegars C: Food Chemistry vinegars and the mature vinegars are the most favorite vinegars in China The aromatic vinegars. .. degradation by quercetin and anthocyanin compounds, compounds that determine onion bulb colors (Kim and others 2004) Red onions contain anthocyanins and quercetin, yellow onions contain quercetin, and
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