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Role of Glassy State on Stabilities of Freeze-Dried Probiotics

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R: Concise Reviews
1 Journal MSP No. No. of pages: 5 PE: Amanda

in Food Science
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Role of Glassy State on Stabilities of Freeze-Dried
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6 Probiotics
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8 Chalat Santivarangkna, Mathias Aschenbrenner, Ulrich Kulozik, and Petra Foerst
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11
Abstract: High viability of dried probiotics is of great importance for immediate recovery of activity in fermented foods
12
and for health-promoting effects of nutraceuticals. The conventional process for the production of dried probiotics is
13
14
freeze-drying. However, loss of viability occurs during the drying and storage of the dried powder. It is believed that
15 achieving the “glassy state” is necessary for survival, and the glassy state should be retained during freezing, drying, and
16 storage of cells. Insight into the role of glassy state has been largely adopted from studies conducted with proteins and
17 foods. However, studies on the role of glassy state particularly with probiotic cells are on the increase, and both common
18 and explicit findings have been reported. Current understanding of the role of the glassy state on viability of probiotics is
19 not only valuable for the production of fermented foods and nutraceuticals but also for the development of nonfermented
20 functional foods that use the dried powder as an adjunct. Therefore, the aim of this review is to bring together recent
21 findings on the role of glassy state on survival of probiotics during each step of production and storage. The prevailing state
22 of knowledge and recent finding are discussed. The major gaps of knowledge have been identified and the perspective of
23
ongoing and future research is addressed.
24
25 Keywords: freeze drying, glass transition, lactic acid bacteria, probiotics
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27
28
29
30
31 Introduction glass can inhibit diffusion and slow down deleterious reactions or
Research and interest regarding probiotics is receiving more changes in the structures and chemical composition. The role
32
focus than ever before. For example, in 2008, more than of glassy state on the functionalities, bioactivity, and stability of
33
34 1000 articles and reviews were published on the subject and more enzymes, pharmaceutical proteins, and foods has been widely re-
35 than 2000 probiotic products launched (Jankovic and others 2010). ported. Nevertheless, current studies on the role of glassy state,
36 The global market for probiotic products was worth US$16 billion particularly with probiotics, are on the increase and both com-
37 in 2008, and the estimated target is a total of US$19.6 billion in mon and explicit findings have been found. It is an aim of this
38 sales in 2013 (Granato and others 2010). For food applications, review to bring together recent findings on the role of glassy state
39 probiotics are mainly employed alone or together with starter on survival of probiotics during each step of the production and
40 cultures in fermented dairy products (for example, yogurts and of the storage period. The prevailing state of knowledge related to
41 cultured drinks). In addition, probiotics are also potentially used the role is revisited, and recent finding is discussed.
42 as nutraceuticals and dry adjunct in nonfermented and nondairy
43 products, such as fruit juices, cereals, dried powder foods (Rivera- Glassy State
44 Espinoza and Gallardo-Navarro 2010). In either case, high vi-
The glassy state is referred to an amorphous metastable state
45 ability of probiotics is of great importance in order to ensure
that resembles a solid but without any long-range lattice order,
46 immediate recovery of fermentation activity (especially Direct Vat
that is, the position of molecules relative to another is more ran-
47 Inoculation-DVI cultures) and to meet the minimal requirements
dom. In other words, it has a solid characteristic/appearance but a
48 for health-promoting effects.
molecular arrangement that is more typical for liquids. A glass has
49 The standard process for the production of dry probiotics is
an extremely high viscosity (for example, typically ≥ 1012 Pa s)
50 freeze-drying. The typical freeze-drying process consists of 3 steps:
and shows temperature-dependent transition (Slade and Levine
51 freezing, primary drying, and secondary drying. During these
1991). When a glass is heated above a certain temperature, the
52 3 steps, cells are exposed to various stresses, especially dehydra-
molecules gain translational mobility and enter a liquid-like state.
53 tion, compromising cell survival. A further survival reduction oc-
The transformation of solid- to liquid-like state is known as glass
54 curs during storage of the dried powder, where the key storage
transition. The most common parameter describing the glassy state
conditions such as relative humidity and temperature play a major
55 and its transition is the glass transition temperature (Tg or Tg  for
56 role. In addition to physiological states of cells, the physical state
a maximally freeze-concentrated solution), below which mate-
57 of the surrounding sample matrix is believed to be critical for sur-
rials exhibit the extremely high viscosity. At T < Tg , diffusion
58 vival of probiotics. The high viscosity of a surrounding amorphous
limited deterioration reactions are inhibited because water in the
59 amorphous glass is immobilized and unavailable.
60 In various studies, sugars or strong glass-forming polymers have
61 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,
62
Q1 Technische Universität München, Germany. Direct inquiries to author Foerst (E-mail: is based on the observation that many anhydrobiotes accumulate
63 petra.foerst@wzw.tum.de). large amount of sugars, especially trehalose and sucrose, inside the
64 cells, for example, 25% of DW (Buitink and Leprince 2004). The


C 2011 Institute of Food Technologists
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doi: 10.1111/j.1750-3841.2011.02347.x Vol. 00, Nr. 0, 2011 r Journal of Food Science R1

Further reproduction without permission is prohibited
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1 Glassy state of dried probiotics . . .
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3 presence of sugars on both sides of the cells was considered being is essential for the survival during freezing (Pehkonen and others
4 necessary for protection against freezing (Duong and others 2006) 2008). Given Tg  of skim milk (–50 ◦ C), sucrose (–46 ◦ C), and tre-
5 and dehydration (Tymczyszyn and others 2007). Crowe and others halose (–40 ◦ C), which are the most common suspending medium
6 (1987) found that intra- and extracellular sugars provide protection used in many studies, and Tg  of pure water (–135 ◦ C), the maxi-
7 of proteins and membranes by forming a glassy matrix that is able mally freeze-concentrated matrix should be obtained by common
8 to interact via hydrogen bonding. This protective effect of sugars industrial freezing protocols, for example, immersion in liquid N2
9 on biomolecules was reported to appear only in case of amor- (–196 ◦ C). Otherwise, the freezing temperature should be selected
10 phous carbohydrate glasses, but not in case of crystalline sugars with care to ensure a maximally freeze-concentrated state and for-
11 (Pikal and Rigsbee 1997; Izutsu and Kojima 2002). In contrast to mation of a glassy structure of the unfrozen continuous phase of
12 the drying of pharmaceutically relevant proteins, where the sensi- the cell suspension. In freeze-drying formulations, cryoprotectants
13 tive biomolecules are directly embedded in the amorphous sugar are used to forestall cell injury. Some viscous cryo-protectants (for
14 glass, several drying-sensitive components of bacterial cells are sit- example, glycerol, sugars, and polymers) increase the viscosity of
15 uated in the intracellular space. Therefore, the protectant must be the freeze-concentrated solution or cytoplasm depending on their
16 transported through the cell membrane. Thus, many high MW permeability. Therefore, the glassy state can be reached at a lower
17 polymers that show limited transportation into cells or in pene- cooling rate and a higher freezing temperature (Morris and others
18 tration and interaction with the phospholipid headgroups of the 2006).
19 cell membrane are not very effective in protecting dried probiotics The annealing step is commonly used for the freeze-drying
20 Q2 (Semyonov and others 2010). of proteins and pharmaceuticals. However, it is not applied for
21 According to Potts (1994), it is still not clear if bacterial glasses the production of freeze-dried probiotics. Annealing is a pro-
22 exist. The DSC technique, which is commonly used for the de- cess, where frozen samples are kept isothermally at a temperature
23 tection of glass transition, is insensitive for cells, where changes in between Tg  and the onset of melting temperature. During anneal-
24 heat capacity are small and or when many transitions occur in the ing, freezable water entrapped in amorphous regions (due to rapid
25 same temperature range. For example, Fonseca and others (2001) freezing) is crystallized into larger and more uniform ice crystals.
26 showed that the Tg of washed cells of L. bulgaricus cannot be de- This is especially true for the frozen probiotics, which are com-
27 Q3 tected. Furthermore, Cerrutti and others (2000) and Fonseca and monly produced by rapid freezing using liquid nitrogen. A pellet
28 others (2001) indicated that the reported Tg is mainly obtained of 2 mm diameter is cooled from 0 to –50 ◦ C in approximately
29 from the added solutes or the drying matrix. However, Hoekstra 10 s or at a freezing rate of approximately 300 ◦ C/min (Oetjen and
30 and others (2001) and Sun and Leopold (1997) showed that the Haseley 2003). The increased content and uniform ice formation
31 state of the cytoplasm is considered to be of crucial importance improve the efficacy of subsequent drying process. The knowledge
32 Q4 for the dehydration tolerance in organisms. Thus, the glassy state of the influence of annealing on probiotics’ survival and stability is
33 of dried cells (if there is any) needs to be considered carefully. very limited. In a study by Ekdawi-Sever and others (2003), it was
34 reported that the annealing does not cause cell death, but improves
35 Role of Glassy State during Freezing storage stability and reduces browning reaction of L. acidophilus.
36 The typical freeze-drying or lyophilization process consists of
37 3 steps: freezing, primary drying or sublimation, and secondary Role of Glassy State during Drying
38 drying or desorption. The ice crystals formed during freezing It is the current opinion that the product temperature during
39 determine the morphology and distribution of the pores (macro- removal of ice crystals should not be higher than the critical tem-
40 scopic cake structure) that are formed during the removal of ice perature. This critical temperature commonly refers to Tg  (or Tg
41 crystals. Thus, the freezing process significantly determines the after sublimation). In this respect, the processing conditions, (for
42 drying behavior of the sample throughout the following process example, pressure and shelf temperature during sublimation) must
43 steps. In order to ensure rapid drying and to facilitate vapor migra- be controlled so that the product temperature is not higher than
44 tion during drying, ice crystals in the suspension should be large Tg  during drying. For this reason, solutes with high Tg are often
45 and contiguous. On the one hand, large ice crystals can only be added to increase Tg  so that the drying can be carried out at a
46 achieved by relatively slow freezing rates (not with liq. N2 ). On higher temperature, and a high Tg of final dried products is ob-
47 the other hand, these large ice crystals can induce cell damage tained to provide a high storage stability. Disaccharide sugars and
48 due to mechanical stress. Generally, slow freezing may accompany oligomeric sugars are preferred as additives for freeze-drying not
49 the eutectic crystallization of buffer salt components, which is also only because they exhibit a higher Tg (Adams and Ramsay 1996),
50 suspected to cause membrane damage (Morgan and others 2006). but also because they can be easily vitrified (Franks 1998; Ward and
51 In other words, optimum freezing conditions often require a com- others 1999). For sugar alcohols such as mannitol, caution must
52 promise between the requirements of the bacteria and the drying be exercised. Mannitol can easily separate from a frozen solution
53 performance. For the production of probiotics, frozen granules are in the form of a crystalline phase (Adams and Ramsay 1996; Kim
54 commonly produced by distributing the cell suspension through and others 1998), resulting in a loss of the product stability after
55 a droplet disc with pores or through a nozzle into liquid N2 . In freeze-drying (Izutsu and others 1994; Izutsu and Kojima 2002).
56 addition to direct freezing in liquid N2 , innovative rapid freezing This reason has been proposed for the little or no protection con-
57 methods were explored by Volkert and others (2008). The frozen ferred by mannitol on freeze-dried malolactic cultures (Zhao and
58 granules are produced by the spray freezing, where cell suspen- Zhang 2005).
59 sion is sprayed in an air blast freezer to get droplets (for example, Recently, “collapse temperature” which is defined as the max-
60 size of ca. 5 to 30 μm). For a frozen solution, at Tg  ,the high imum temperature preventing the structure of the dried product
61 viscosity of the unfrozen phase will inhibit ice crystal formation; from macroscopic collapse (Tc ) (Fonseca and others 2004a) was
62 and therefore, a maximally freeze-concentrated solution is formed proposed to use as the critical temperature for freeze-drying of
63 (Roos 1997). It has been suggested that the formation of a max- drying formulation with cells. In comparison, Tc and Tg  are mea-
64 imally freeze-concentrated matrix with entrapped microbial cells sured with techniques based on different principles. DSC has been

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3 commonly used over decades to determine the Tg as a mid- or dition where the difference between product and glass transition
4 onset point of the temperature range that the endothermic shift in temperature was so high that the sample showed a collapsed struc-
5 heat capacity appears. The measurement of Tg  is carried out with ture. The results are in agreement with unexpected results from a
6 a representative frozen sample ex situ at atmospheric pressure. In recent study by Schersch and others (2010) with pharmaceutically
7 contrast, Tc is commonly determined by a freeze-drying micro- relevant proteins. Loss of biological activities of the proteins was
8 scope as the visual structural collapse during the simulated subli- not observed in collapsed and noncollapsed cakes. The collapsed
9 mation of ice crystals under a set vacuum level. In other words, Tc lyophilizate, the appearance of which is unacceptable for pharma-
10 reflects the physical state of frozen matrix during drying, whereas ceutial products, is not critically important for probiotic products.
11 Tg  rather reflects the physical state regardless of the drying condi- The dried lyophilizate has to be milled into powder to be mixed
12 tions. The state diagram showing glass transition temperature and with other food components, mixed with other fillers for probi-
13 collapse temperature is depicted in Figure 1 (adapted and modified otic tablets or capsules, as well as used as starter cultures. When
14 from Roos 2010). In the absence of cells, the difference between the collapsed structure as a result of drying does not negatively af-
15 Tg  and Tc is very small. Although the presence of cells does not fect the viability of cells, the proposed concept “collapse drying,”
16 clearly influence Tg (Fonseca and others 2001; Schoug and oth- which is expected to substantially reduce drying time (Schersch
17 ers 2006), it significantly increases Tc (Fonseca and others 2004a). and others 2010) can be possibly applied for the production of
18 Bacteria can give some kind of structure and thus reduce or avoid freeze-dried probiotics.
19 viscous flow when Tg of pure sugar solution is reached. The in- In addition to free water, which is removed during primary
20 crease depends on the cell types, that is, size, shapes, and cell chain drying, cells contain ca. 0.25 g water/g dry weight of strongly
21 formation and concentration (Fonseca and others 2004b). As a re- associated water that is unfreezable. This strongly associated water
22 sult, a freeze-drying matrix with cells is more robust, and when Tc is removed by desorption. The shelf temperature is elevated to
23 is taken as the critical temperature, it allows the drying stage with promote desorption of water. At the end of the secondary dry-
24 a higher product temperature (or practically drying temperature). ing, the sample should have a water content that is optimal for
25 This is of economical importance because it is estimated that the storage. It has been suggested that the moisture content of 1%
26 increase in a degree of product temperature will decrease primary or less is required for a long-term shelf life (Nakamura 1996);
27 drying time by about 13% (Tang and Pikal 2004). however, Gardiner and others (2000) showed that moisture re-
28 Nevertheless, the opinion that cells should be retained in glassy moval below 4% may be considered practically enough. It was
29 state during drying lacks clear empirical evidence. The physical also reported in a study by Zayed and Roos (2004) that the opti-
30 state has been measured mostly by analyzing the frozen and dry mal moisture content for storage of freeze-dried L. salivarius ssp.
31 sample before and after drying, while the physical state during salivarius is in the range of 2.8% to 5.6%. Therefore, it is still
32 drying is changed with drying time and conditions. In our re- debatable whether cells should be dried to moisture content as
33 cent study (Foerst and others 2010), several drying protocols were low as possible. An argument is that lipid oxidation is enhanced
34 carried out, and the viability was considered in relation to physi- at very low water contents, and water acts as a protectant against
35 cal state, residence time in rubbery state and moisture content of oxidation.
36 samples. The study showed that glassy state may not play a signif-
37 icant role on viability of cells during drying step of freeze-drying. Role of Glassy State during Storage
38 The survival decreased similarly for conditions where the samples Low moisture contents of dried cells after drying do not guaran-
39 remained in glassy state for the whole part of drying, and for con- tee high stability during storage. The moisture is not constant, and
40
41
42
Figure 1–State diagram showing different
43
regions and state of a drying matrix: Tm , onset
44 
of ice melting; Tm , onset of ice melting in a
45 maximally freeze-concentrated solute matrix;

46 Tg , glass transition temperature; Tg , glass
47 transition temperature of the maximally
freeze-concentrated solute matrix; Tgw , glass
48 transition of water, and Tc , collapsed
49 temperature. Tc approximate to Tg and


50 increases in the presence of cells. The shaded

51 area shows a temperature range for maximum

ice formation for the solute concentration Cg .
52
The glass line indicates the glass transition
53 temperature at various solute concentrations
54 (adapted and modified from Roos 2010).
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3 desorption, adsorption, and the glass transition may occur during L. coryniformis by Schoug and others (2010) and with L. rhamnosus
4 storage depending on the specific combination of atmospheric by Miao and others (2008) where viability of cells clearly de-
5 relative humidity and storage temperature. Equilibrium of ambi- creased at T – Tg higher than –50 ◦ C and ca. –30 ◦ C, respectively.
6 ent conditions may only take a few hours or days (Kurtmann and Furthermore, the glassy state does not play major protective role
7 others 2009b) or as long as to 1 to 2 mo (Fonseca and others against major storage deteriorative reactions such as lipid oxidation
8 2001). This equilibration period is often not taken into account (Andersen and others 1999; Kurtmann and others 2009a), because
9 in studies with the storage stability and Tg . At a critical storage the free radical reaction is not diffusion limited. In addition, lipid
10 temperature Tg , the critical water content and storage relative hu- carbonyls as formed as secondary oxidation products from lipid ox-
11 midity (or aw ) can be determined when information on sorption idation have been suggested to participate in browning reactions
12 isotherm of cells is available (Figure 2 adapted and modified from through reactions with amino groups in Maillard-like reactions.
13 Fonseca and others 2001). Information on the sorption isotherm Browning reaction is related to viability loss in freeze-dried cells
14 of dried probiotics is necessary to avoid inactivation due to mois- (Kurtmann and others 2009c). It was suggested also that the na-
15 ture gain during storage. Furthermore, it can be used to corre- ture of the sugar was more important for storage stability than the
16 late the storage relative humidity and the final moisture content physical state of the matrix with the nonreducing sucrose provid-
17 of dried cells to avoid unnecessary drying time from drying of ing better stability than the reducing lactose (Kurtmann and others
18 cells far beyond the moisture content corresponding to storage 2009b).
19 aw . However, in comparison to foods, very little information on Alternatively, it was also proposed that a biomaterial is most sta-
20 sorption isotherm of probiotics is available. Notably, stability of ble at or below its monolayer moisture content or aw , which varies
21 probiotics is commonly considered with respect to survival. The in each biomaterial and with environmental conditions (Pitombo
22 role of glassy state has never been studied in correlation to probiotic and others 1994). For example, the monolayer moisture con-
23 properties, although the properties are vital for probiotic products tent of L. bulgaricus (Fonseca and others 2001) and encapsulated
24 and there is a report about change in the properties during stor- L. rhamnosus (Ying and others 2010) was 10% and 3%, respectively.
25 age. The amount of bacteriocin production in freeze-dried vaginal This concept cannot completely explain the storage stability of
26 lactobacillus strains was significantly reduced after storage for 12 mo, dried cells as well, considering that an increase in temperature sig-
27 whereas the production of other antimicrobial substances (lactic nificantly affected the inactivation rate but minimally affected the
28 acid and hydrogenperoxide) and the auto-aggregation were still water activity. Moreover, it provides only information on strongly
29 retained (Juarez Tomas and others 2009). associated and free water but does not indicate when the molecu-
30 Nevertheless, recent studies on storage of dried probiotics cast lar mobility starts to increase. A strong combined effect of water
31 doubt upon the hypothesis that chemical reactions are completely activity and temperature was reported and it was suggested that
32 halted in the glassy state. It does not solely determine the storage aw and Tg are coupled (Kurtmann and others 2009b). Unlike
33 stability of dried cells, and inactivation still occurs during storage freeze-dried proteins, a bacterial cell is not a unique entity but
34 at a temperature T – Tg = 0 (Higl and others 2007; Kurtmann contains the outside compartment, which is mainly drying ma-
35 and others 2009b). It was suggested that in order to achieve nearly trix and the inside compartment, which contains cell components
36 complete reduction of molecular movements, amorphous phar- with different affinities to water. Hence, not all components or
37 maceutical solids should be stored 50 ◦ C below Tg (Hancock and functions of cells are equally sensitive to moisture or temperature.
38 others 1995). Similar findings were reported also in a study with In other words, there are systems where the inactivation is strongly
39
40
41
Figure 2–Relationships between glass transition
42 temperature, water content, and water activity.
43 At critical storage temperature a (Tg ), the
44 according storage relative humidity (aw ) and
45 water content of a sample are b and c (adapted
and modified from Fonseca and others 2001).
46
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3 dependent on temperature and others that are strongly depend on Izutsu K, Kojima S. 2002. Excipient crystallinity and its protein-structure-stabilizing effect during
4 aw (Higl and others 2007). freeze-drying. J Pharm Pharmacol 54:1033–9.
Izutsu K, Yoshioka S, Terao T. 1994. Effect of mannitol crystallinity on the stabilization of
5 enzymes during freeze-drying. Chem Pharm Bull (Tokyo) 42:5–8.
6 Conclusion and Outlook Jankovic I, Sybesma W, Phothirath P, Ananta E, Mercenier A. 2010. Application of pro-
biotics in food products-challenges and new approaches. Curr Opin Biotechnol 21:
7 Despite the current opinion that a glassy state is highly im- 175–81.
8 portant for the stability of freeze-dried biomaterial and thus has Juarez Tomas MS, Bru E, Martos G, Nader-Macias ME. 2009. Stability of freeze-dried vaginal
Lactobacillus strains in the presence of different lyoprotectors. Can J Microbiol 55:544–52.
9 to be maintained throughout the whole production process and Kim AI, Akers MJ, Nail SL. 1998. The physical state of mannitol after freeze-drying: effects of
10 storage, no study could empirically confirm its decisive effects, mannitol concentration, freezing rate, and a noncrystallizing cosolute. J Pharm Sci 87:931–5.
Kurtmann L, Carlsen CU, Risbo J, Skibsted LH. 2009a. Storage stability of freeze-dried Lacto-
11 especially in case of probiotics. This can be due to the fact that bacillus acidophilus (La-5) in relation to water activity and presence of oxygen and ascorbate.
12 unlike proteins and foods, dried probiotic cells are largely still Cryobiol 58:175–80.
Kurtmann L, Carlsen CU, Skibsted LH, Risbo J. 2009b. Water activity-temperature state di-
13 intact and actually separated from the drying matrices. Thus, agrams of freeze-dried Lactobacillus acidophilus (La-5): influence of physical state on bacterial
14 in case of probiotics, common results relate to the physical state of survival during storage. Biotechnol Prog 25:265–70.
15 the surrounding drying matrices regardless of the actual physical
Kurtmann L, Skibsted LH, Carlsen CU. 2009c. Browning of freeze-dried probiotic bacteria
cultures in relation to loss of viability during storage. J Agric Food Chem 57:6736–41.
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Vol. 00, Nr. 0, 2011 r Journal of Food Science R5

R: Concise Reviews
in Food Science

Queries

Q1 Author: Please provide ZIP code for the affiliation.

Q2 Author: Please provide the expanded form of “MW.”
Q3 Author: Please provide genus name for the scientific term “L. Bulgaricus.”
Q4 Author: Please provide the expanded form of “DSC.”
Q5 Author: Please provide volume number and page range for reference Foerst and others (2010).
Q6 Author: Please provide publisher location for references Nakamura (1996) and Oetjen and Haseley (2003).

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