Influence of acid hydrolysis and dialysis of kcarrageenan on its ice recrystallization inhibition activity

The objective of this study was to investigate the influence of molecular weight of kcarrageenan on its ice recrystallization inhibition (IRI) activity. To reduce the molecular weight, acid hydrolysis with 0.1 M trifluoroacetic acid (TFA) at 80 C or 0.1 M hydrochloric acid (HCl) at 60 C was performed. In addition, molecular weight was reduced by dialyzing kcarrageenan against demineralized water. It was demonstrated that IRI activity of kcarrageenan decreases with decreasing molecular weight, which is contrary to previous studies. It was shown in our study that the different results may be attributed to an aggregation of kcarrageenan molecules due to high NaCl concentration, originated from HCl and subsequent neutralization with NaOH. This aggregation causes a decrease in IRI activity. Furthermore, it was shown that the addition of a small amount of NaCl can lead to an increase in IRI activity of kcarrageenan

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In ﬂuence of acid hydrolysis and dialysis of k -carrageenan on its ice

recrystallization inhibition activity

a KIT (Karlsruhe Institute of Technology), Institute of Process Engineering in Life Sciences, Section I: Food Process Engineering, Kaiserstrasse 12, 76131

Karlsruhe, Germany

b KIT (Karlsruhe Institute of Technology), Department of Food Chemistry and Phytochemistry, Institute of Applied Biosciences, Adenauerring 20a, 76131

Karlsruhe, Germany

a r t i c l e i n f o

Article history:

Received 2 December 2016

Received in revised form

7 April 2017

Accepted 11 April 2017

Available online 12 April 2017

Keywords:

Acid hydrolysis

Dialysis

Ice recrystallization inhibition

Ions

k-Carrageenan

Molecular weight

a b s t r a c t

The objective of this study was to investigate the inﬂuence of molecular weight ofk-carrageenan on its ice recrystallization inhibition (IRI) activity To reduce the molecular weight, acid hydrolysis with 0.1 M triﬂuoroacetic acid (TFA) at 80C or 0.1 M hydrochloric acid (HCl) at 60C was performed In addition, molecular weight was reduced by dialyzing k-carrageenan against demineralized water It was demonstrated that IRI activity ofk-carrageenan decreases with decreasing molecular weight, which is contrary to previous studies It was shown in our study that the different results may be attributed to an aggregation ofk-carrageenan molecules due to high NaCl concentration, originated from HCl and sub-sequent neutralization with NaOH This aggregation causes a decrease in IRI activity Furthermore, it was shown that the addition of a small amount of NaCl can lead to an increase in IRI activity ofk-carrageenan

1 Introduction

Freezing is a popular method for food preservation It extends

the shelf life by slowing down chemical and enzymatic reactions as

well as microbial reproduction (Gaukel, 2016) However, during

storage and distribution of frozen products, especially under

un-favorable temperature conditions, changes of ice crystal

micro-structure can affect the quality of frozen food products These

changes are termed as “recrystallization” which is deﬁned as

changes in size, number, and shape of unique ice crystals while

keeping the total volume of ice constant (Cook and Hartel, 2010)

Recrystallization processes have a high impact on the appearance

and on the texture of frozen food (Pham and Mawson, 1997) For

instance, ice cream becomes coarse and unacceptable if ice crystals

grow during storage and exceed a threshold detection size (Hartel,

1996, 2001) Recrystallization processes can be retarded by low and

constant storage temperatures (Donhowe and Hartel, 1996) or by

formulation, especially by the addition of ice recrystallization

in-hibitors Traditionally, hydrocolloids are added to inhibit

recrystallization in ice cream (Adapa et al., 2000; Bahramparvar and Mazaheri Tehrani, 2011; Marshall et al., 2003; Miller-Livney and Hartel, 1997) However, despite a large amount of research, the exact recrystallization inhibition mechanism of hydrocolloids is still not understood in every detail (Bahramparvar and Mazaheri Tehrani, 2011; Leiter and Gaukel, 2016)

One such hydrocolloid is k-carrageenan which is a linear, sulfated polysaccharide extracted from certain species of red sea-weeds (Rhodophyceae) (McHugh, 2003) It is composed of an alternating disaccharide unit of 1,3-linkedb-D-galactose-4-sulfate and 1,4-linked 3,6-anhydro-a-D-galactose (Rochas and Rinaudo,

1984) The weight average molecular weight of commercially available k-carrageenan ranges typically from 300 to 650 kDa (Hoffmann et al., 1996; Lahaye, 2001; Piculell, 2006; Prajapati et al.,

2014) However,k-carrageenan is quite polydisperse Thus, most of the material is found in the range of 102e103kDa but there is also a long tail on the low-molecular side of the distribution (Piculell,

2006).k-carrageenan can form thermoreversible gels upon cool-ing (Piculell, 2006) Therefore, the polysaccharide is used as gelling, thickening, and stabilizing agent especially in food products and in cosmetics and pharmaceutical formulations (Campo et al., 2009; McHugh, 2003; Rinaudo, 2008; van de Velde and De Ruiter,

2006) The molecular mechanism of gelation is still not

* Corresponding author.

E-mail address: andreas.leiter@kit.edu (A Leiter).

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Journal of Food Engineering 209 (2017) 26e35

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understood in detail A widely accepted model of the gelation

mechanism is the“domain model” (Mangione et al., 2003; Morris

et al., 1980) According to the “domain model”, k-carrageenan

molecules exist in solution as unstructured random coils above a

certain temperature A temperature reduction below this so called

coil-helix transition temperature induces the formation of double

helices Below the transition temperature, the intermolecular

as-sociation between the double helices is limited to the formation of

small independent domains involving a limited number of double

helices However, when cations are present, double helices of

different domains aggregate to enable long-range cross-linking

(Campo et al., 2009; Morris et al., 1980) which can lead to the

for-mation of a gel (Mangione et al., 2003) However, despite a lot of

research in this area, it is still a matter of debate if double or single

helices are formed (Ciancia et al., 1997; Rochas and Rinaudo, 1984;

Schefer et al., 2015; Smidsrød and Grasdalen, 1982; Ueda et al.,

1998; Viebke et al., 1995)

In addition to its ability to form a gel, k-carrageenan shows

strong ice recrystallization inhibition (IRI) activity in frozen sucrose

solutions (Chun et al., 2012; Gaukel et al., 2014;

Kaminska-Dworznicka et al., 2015; Leiter et al., 2017) Although several

po-tential mechanisms for IRI activity of hydrocolloids have been

suggested, the exact IRI mechanism ofk-carrageenan is still not

understood It is assumed that the IRI activity of certain

hydrocol-loids originates from a decrease in mobility of water molecules due

to water binding or steric hindrance (Caldwell et al., 1992;

Miller-Livney and Hartel, 1997; Regand and Goff, 2002, 2003) However,

by using pulsedﬁeld gradient (PFG) nuclear magnetic resonance

(NMR) it was demonstrated that the addition ofk-carrageenan to a

sucrose solution does not signiﬁcantly inﬂuence the self-diffusion

coefﬁcient of water (Gaukel, 2004) Furthermore, the formation of

a gel is often discussed as a possible IRI mechanism of hydrocolloids

(Bahramparvar and Mazaheri Tehrani, 2011; Goff et al., 1999;

Regand and Goff, 2003) However, investigations on the relation

of gelation and IRI activity ofk-carrageenan showed that the

for-mation of ak-carrageenan gel results in a reduction of its IRI activity

(Leiter et al., 2016a) Moreover, it is assumed that certain

hydro-colloids may retard the incorporation of water molecules into the

ice crystal lattice by modifying the ice crystal/liquid interface

(Martin et al., 1999) or by binding to the ice crystal surface (Gaukel

et al., 2014; Martin et al., 1999; Sutton et al., 1996, 1997) In previous

studies, it was shown that ice crystals in ak-carrageenan sucrose

solution are more rectangular and elongated similar to ice crystal

shapes found in sucrose solutions with ice-binding proteins (IBP)

(Gaukel et al., 2014) In addition, it was shown that in solutions,

wherek-carrageenan exhibits IRI activity, more angular ice crystals

are present, in contrast to rather round ice crystals in solutions,

wherek-carrageenan exhibits no IRI activity (Leiter et al., 2017)

Therefore, it has been suggested that the IRI activity ofk

-carra-geenan originates from an interaction with the ice crystal surface

similar to IBPs or polyvinyl alcohol (PVA) (Budke and Koop, 2006;

Davies, 2014; Inada and Lu, 2003)

In a previous study we showed that IRI activity ofk-carrageenan

was signiﬁcantly increased at pH 1 adjusted by hydrochloric acid

(Leiter et al., 2017) The increase in IRI activity was discussed to be

possibly attributed to a reduction in molecular weight due to

hy-drolysis This led to the hypothesis that smaller k-carrageenan

molecules exhibit a higher IRI activity.Kaminska-Dworznicka et al

(2015)also showed a signiﬁcant increase in IRI activity when the

average molecular weight of k-carrageenan was reduced from

34,000 kDa to 2700 kDa by acid hydrolysis using hydrochloric acid

However, the considerably higher weight average molecular weight

in this study compared to the typical molecular weight of untreated

k-carrageenan (300e650 kDa) suggests aggregation of k

-carra-geenan molecules Aggregation possibly occurred due to the

presence of Naþcations by neutralization with 0.1 M NaOH or due

to a high ion content of the raw material In our previous papers (Leiter et al., 2016a, 2017), we already showed that aggregation ofk -carrageenan helices is induced by the presence of cations, which leads to a reduction of IRI activity Thus, it is not sure whether the increased IRI activity, which was observed by Kaminska-Dworznicka et al (2015), is really due to an increased IRI activity

of hydrolyzedk-carrageenan molecules or just due to a reduction of aggregates

Therefore, the objective of this study was to investigate if there

is an increased IRI activity of k-carrageenan molecules with reduced molecular weight For this purpose, k-carrageenan was hydrolyzed by different methods and reduction of molecular weight was verified by size-exclusion chromatography (SEC) before IRI experiments Hydrolysis methods were adjusted to avoid additional salt in thek-carrageenan samples Thus, IRI experiments should not be influenced by aggregation ofk-carrageenan mole-cules Firstly,k-carrageenan was hydrolyzed by 0.1 M trifluoroacetic acid (TFA), because TFA is volatile and evaporates during freeze-drying Therefore, no subsequent neutralization of the acid is necessary and no ions are added to the polysaccharide preparation Secondly, molecular weight was reduced by dialyzing k -carra-geenan against demineralized water and subsequent freeze-drying This process was described to induce autohydrolysis of the k -carrageenan (Hoffmann et al., 1996) For the examination of dif-ferences between the results ofKaminska-Dworznicka et al (2015) and the results provided in this study,k-carrageenan was also hy-drolyzed with 0.1 M hydrochloric acid (HCl) and neutralized with NaOH In addition, rheological temperature sweep measurements were performed to detect possible aggregation of k-carrageenan molecules

2 Materials and methods 2.1 Materials

k-carrageenan was used as unstandardized extract from the red seaweed Eucheuma Cottonii produced by gel-press method in the Philippines It was provided by Eurogum A/S (Herlev, Denmark) and was used in this study without further puriﬁcation Ion (major element is potassium with a mass fraction of 7.29%) and sulfate content (19.55%) of the unstandardized sample is described in previous papers (Leiter et al., 2016a, 2017) Because the composi-tion of naturalk-carrageenan may differ from batch to batch (van

de Velde et al., 2002), k-carrageenan from the same batch was used for all experiments Sucrose was common household sugar from a local supplier All other chemicals were of analytical grade 2.2 Preparation of differentk-carrageenan samples

2.2.1 Dialysis

k-Carrageenan solution (1 mg mL1) was prepared by dissolving thek-carrageenan extract in pure water at 60C for 30 min An aliquot (50 mL) of thisk-carrageenan solution was dialyzed 36 h against 3 L of demineralized water using a dialysis tubing (Carl Roth, Germany) with a molecular weight cut-off of 3500 Da The demineralized water was replaced every 12 h Subsequently, the dialyzedk-carrageenan solution was freeze-dried

For an optimal comparison of the IRI activity of the dialyzedk -carrageenan with the untreatedk-carrageenan, the same ion con-centration has to be present in both solutions Therefore, a second dialyzed k-carrageenan sample, which has the same ion concen-tration as the untreated k-carrageenan, was prepared For this purpose, the polysaccharide solution was treated as described above, but the dialysis tubing was placed in 3 L of 2.5 mM KCl

A Leiter et al / Journal of Food Engineering 209 (2017) 26e35 27

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solution after the dialysis against demineralized water to add Kþ

cations to the dialyzedk-carrageenan before freeze-drying The KCl

solution was replaced every 12 h for 36 h The concentration of

2.5 mM KCl is the same as the total salt concentration in a

1 mg mL1 k-carrageenan extract solution (Leiter et al., 2016a,

2017) To obtain a sufﬁcient amount of freeze-dried sample for

the following experiments, this procedure was repeated several

times and the freeze-dried samples were mixed and stored in a

desiccator

2.2.2 Acid hydrolysis ofk-carrageenan with triﬂuoroacetic acid

k-carrageenan stock solutions (2 mg mL1) for acid hydrolysis

were prepared by dissolvingk-carrageenan extract or dialyzedk

-carrageenan (each 400 mg) in 200 mL of pure water at 60C for

30 min After cooling down to room temperature, 10 mL of the stock

solution was mixed with 10 mL of 0.2 M triﬂuoroacetic acid (TFA) to

get aﬁnal TFA concentration of 0.1 M The 0.1 M TFAk-carrageenan

solution was heated to 80C and kept at this temperature for 5 min,

10 min, 20 min or 60 min The hydrolysates were cooled down

immediately by placing the solutions in ice water The conditions of

the TFA hydrolysis should cleave some glycosidic bonds, but only

negligible degradation of 3,6-anhydro-a-D-galactose units

(Stevenson and Furneaux, 1991; Yang et al., 2009)

To obtain a sufﬁcient amount of each sample, the method was

performed with several aliquots of the stock solution Finally, the

aliquots of same TFA treatment times were mixed, freeze-dried and

stored in a desiccator

2.2.3 Acid hydrolysis ofk-carrageenan with hydrochloric acid

For HCl hydrolysis, 10 mL of the stock solution was mixed with

10 mL of 0.2 M HCl to get aﬁnal HCl concentration of 0.1 M The HCl/

k-carrageenan solution was heated to 60C and kept at this

tem-perature for 30 min The hydrolysate was cooled down immediately

by placing the solution in ice water Just as for the acid hydrolysis

with TFA, these hydrolysis conditions should cleave some glycosidic

bonds, but negligible degradation of 3,6-anhydro-a-D-galactose

units (Sun et al., 2015) To avoid further hydrolysis, the solution was

adjusted to pH 7 with 0.1 M NaOH before freeze-drying In the

results section, this freeze-dried sample is designated as sample B

A non-hydrolyzed referencek-carrageenan sample, which

con-tains the same amount of salt as the hydrolyzed k-carrageenan

sample, was prepared by dissolving 20 mg of k-carrageenan in

20 mL of 0.1 M NaCl solution The pH of this solution was 6

Therefore, the pH was adjusted to 7 with 0.1 M NaOH The

non-hydrolyzed reference sample solution was freeze-dried and

desig-nated as sample A To obtain a sufﬁcient amount of each sample, the

method was performed with several aliquots of the stock solution,

and samples were stored as described in section2.2.2

The calculated mass ratio of NaCl to k-carrageenan in both

sample A and B is 5.844 Thus, theﬁnal NaCl concentration cNaClin a

49% sucrose solution with a speciﬁc concentration cA/Bof sample A

or B can be calculated according to the following equation:

cNaCl ¼ 1cA=B

2.3 Analysis by size exclusion chromatography

Polymer degradation was analyzed by size-exclusion

chroma-tography (SEC) The system consisted of a K-500 pump (Knauer,

Germany), a Degasys-1310 degasser (Knauer, Germany), a

Sephar-ose CL-4B column (bed volume: 85 cm 1.6 cm, GE-Healthcare,

Great Britain), and a refractive index (RI) detector (Smartline

2300, Knauer, Germany) As helix formation and aggregation ofk -carrageenan molecules affect the SEC separation (Ueda et al., 1998; Viebke et al., 1995), the column was heated at 60C to maintaink -carrageenan molecules in the disordered coil form and to prevent aggregation (seeFig 1and section3.1) Sodium phosphate buffer (0.05 M, pH 6) containing 0.2 M NaCl was used as eluent at aﬂow rate of 0.5 mL min1 Samples were dissolved in the mobile phase (1 mg mL1), and the injection volume was 5 mL

A calibration with dextran standards (2000 kDa, 1050 kDa,

400 kDa, 125 kDa, 45 kDa; Fluka, Switzerland) was performed for a rough estimation of the apparent molecular weight of the main peaks Mpof the elution proﬁle The relationship between molecular weight of the main peak Mp(Da) and retention time t (min) fol-lowed equation(2):

2.4 Recrystallization experiments 2.4.1 Sample preparation IRI activity of the different hydrolyzedk-carrageenan samples was studied in a 49% (w/w) sucrose solution prepared with dem-ineralized water For this purpose, the appropriate amount ofk -carrageenan sample (ﬁnal concentration 0.146 mg mL1, 1 mg mL1

or 6.844 mg mL1) was dissolved in the sucrose solution by continuous stirring at 60C for 30 min After dissolving, the solu-tion was cooled down to room temperature

In order to investigate the inﬂuence of NaCl on the IRI activity of

k-carrageenan, the appropriate amount of salt (ﬁnal concentration 0.3 mM, 14.6 mM, 30 mM, 60 mM or 100 mM) was mixed with the

k-carrageenan powder (ﬁnal concentration 1 mg mL1) before adding the sucrose solution Then, thek-carrageenan-salt mix was dissolved in the sucrose solution by continuous magnetic stirring at

60C for 30 min

2.4.2 Sample freezing and storage Sample freezing and storage was performed as previously described byLeiter et al (2016b) For ice recrystallization analysis,

Fig 1 Temperature sweep analysis (2C min1) of untreatedk-carrageenan extract (c k -carr ¼ 1 mg mL 1 ) dissolved in the mobile phase (0.05 M sodium phosphate buffer

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sample solution (18ml) was placed between two microscope cover

slips (0.13e0.16 mm thick) stuck on an object slide The slide was

covered with another cover slip and sealed with silicone The cover

slips were used as spacers to allow ice crystal growth in all

di-mensions After drying of the silicone (1 h), samples were subjected

to fast freezing by immersion in liquid nitrogen for a few seconds

By using this freezing step, the aqueous solution was transformed

into a glassy state After freezing, samples were stored at a constant

temperature of12C± 0.1C in a small externally cooled storage

chamber This procedure allows the samples to crystallize in a

uniform way during equal heating conditions from the glassy state

to the storage temperature In contrast to the temperature of a

typical household freezer (about18C), a storage temperature

of12C was selected to accelerate ice recrystallization processes

and thus reduce the necessary storage time for a IRI

characteriza-tion (Donhowe and Hartel, 1996) Under the described conditions,

resulting ice volume fraction is approximately 22% according to the

sucrose/water phase diagram from Riedel (1949) The storage

chamber itself was placed in a cooled glove box with a temperature

of12C± 1C The temperature inside the storage chamber and

glove box was recorded by thermocouples during the storage time

ofﬁve days

2.4.3 Microscopy and image analysis

During storage time, pictures of ice crystals were taken with a

digital camera (Altra SIS20, Olympus, Japan) attached to a

polari-zation microscope (BX41, Olympus, Japan) installed in the glove

box Pictures were taken 3 h and 96 h after freezing For evaluation

of the pictures, the contour of an ice crystal was manually

cir-cumscribed on a computer with the software ImagePro Insight 9.1

(Media Cybernetics, USA) From the deﬁned area of each ice crystal,

the equivalent diameter was calculated as the diameter of a circle

with the same area To get a representative distribution of ice

crystal sizes, 300 to 400 ice crystals were analyzed per object slide

and the arithmetic equivalent diameter was calculated For each

experiment, two object slides were prepared and each experiment

was performed twice Thus, mean ice crystal diameter x and

stan-dard deviations were determined from the arithmetic equivalent

diameters of four object slides For determination of statistical

signiﬁcance of mean ice crystal diameters One-way ANOVA with

Tukey’s test was carried out (alpha ¼ 0.05)

In this study, the term“IRI activity” ofk-carrageenan is used

when mean ice crystal diameters in sucrose solution with k

-carrageenan are signiﬁcantly smaller than mean ice crystal

di-ameters in a pure sucrose solution at a given storage time In

addition, the smaller the mean ice crystal diameter in a solution

withk-carrageenan, the higher its IRI activity is rated However, this

is only true whenk-carrageenan does not affect ice crystal

nucle-ation To the best of our knowledge, the inﬂuence ofk-carrageenan

on nucleation was not investigated so far, and only limited data are

available for nucleation from the glassy state in food systems

(Hartel, 2001) However, in contrast to nucleation from the glassy

state,Muhr and Blanshard (1986)showed that polysaccharides do

not signiﬁcantly affect ice nucleation in a sucrose solution, when

the system crystallizes from solution phase Thus, even if we cannot

completely exclude thatk-carrageenan affects ice nucleation from

the glassy state, we assume that the initial ice crystal diameters are

the same for all sucrose solutions in this study However, additional

experiments are necessary in the future to prove this assumption

2.5 Rheological temperature sweep measurements

Rheological measurements were performed on a Physica MCR

101 rheometer (Anton Paar GmbH, Austria) with a double gap

ge-ometry (DG 26.7) Temperature was controlled by the temperature

device C-PTD 200 with Peltier temperature control (Anton Paar GmbH, Austria) Coil-helix transition and aggregation was deter-mined by a temperature sweep, which was performed similar as described previously (Leiter et al., 2016a) For temperature sweep analysis, k-carrageenan solutions were prepared as described in section2.4.1and kept at 60C to maintain k-carrageenan in the disordered coil form The hotk-carrageenan solution was loaded in the measuring device preheated to 60C To reduce water evapo-ration, the solution between double gap geometry and outer wall was coated with a thin layer of low viscosity parafﬁn oil After-wards, the solution was held at 60C for 5 min Storage modulus (G’) was determined during cooling (2C min1) to 0C and during reheating (2C min1) to 60C afterﬁve minutes holding time at

0C Temperature sweep was performed at 1 Hz within the linear viscoelastic region All rheological data shown are the mean of two experimental replicates

3 Results and discussion 3.1 Determination of the coil-helix and helix-coil transition temperatures ofk-carrageenan

First, it was evaluated whether a column temperature of 60C during the size-exclusion chromatography (SEC) allows for the analysis ofk-carrageenan in the disordered coil form Hereby, it can

be ascertained that the chromatograms reﬂect singlek-carrageenan molecules instead of k-carrageenan aggregates For this purpose, rheological temperature sweep analysis with untreated k -carra-geenan extract dissolved in the SEC mobile phase was performed

In this experiment, aggregation is usually evident from a thermal hysteresis between the transition temperatures from coil to helix during cooling and from helix to coil during heating (Piculell,

2006) As shown inFig 1, G0increases rapidly at about 20C dur-ing cooldur-ing, which is considered to be due to the coil-helix transi-tion During heating, the decrease of G’, which is considered as the helix-coil transition, is shifted to a higher temperature of about

30C Thus, a thermal hysteresis between the transition tempera-tures and aggregation of k-carrageenan molecules take place However, at a temperature of 60 C,k-carrageenan is in the coil form as the temperature is beyond the helix-coil transition tem-perature of about 45C Thus, it can be concluded that the esti-mated molecular weight at a column temperature of 60 C represents singlek-carrageenan molecules

3.2 Molecular weight distribution and ice recrystallization inhibition (IRI) activity ofk-carrageenan after acid hydrolysis with 0.1 M triﬂuoroacetic acid (TFA)

SEC chromatograms of untreated and hydrolyzedk-carrageenan are shown inFig 2 The hydrolysis with 0.1 M TFA was performed for different time periods For the untreated k-carrageenan, the molecular weight of the main peak Mpis about 1420 kDa according

to equation (2) Besides, there is a typical long tail at the low-molecular side of the distribution, which has already described in literature (Piculell, 2006) The second peak at a retention time of about 370 min is most likely due to the ions, which are already present in the usedk-carrageenan extract (see section2.1) Hy-drolysis with 0.1 M TFA results in a signiﬁcant reduction of mo-lecular weight for all hydrolysis times The main peak of momo-lecular weight distribution is shifted from 1420 kDa to about 20.3 kDa Although the peaks of thek-carrageenan hydrolysates cannot be completely resolved, a continuous reduction of the molecular weight is evident from the chromatograms This is demonstrated by the increasing intensities of the peak at 20.3 kDa and the peak derived from low molecular weight compounds These results

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show that hydrolysis ofk-carrageenan with 0.1 M TFA leads to a

reduction of molecular weight with increasing hydrolysis time

In the next step, the IRI activity of the differentk-carrageenan

hydrolysates was investigated InFig 3, the mean ice crystal

di-ameters in frozen sucrose solutions containing hydrolyzed k

-carrageenan samples after 3 h and 96 h storage time are depicted

In addition, the mean ice crystal diameters in a sucrose solution

with and without untreatedk-carrageenan are shown

First of all, it can be concluded that recrystallization occurs in all

solutions as indicated by an increase of the mean ice crystal

di-ameters over storage time for all samples This is, as expected,

mostly distinct in the sucrose solution without added k

-carra-geenan At both storage times, the mean ice crystal diameter in a

solution with untreatedk-carrageenan is signiﬁcantly smaller than

the mean ice crystal diameter in pure sucrose solution, showing the

strong IRI activity of untreatedk-carrageenan After a storage time

of 3 h only hydrolysis with 0.1 M TFA for 60 min leads to a

significantly larger mean ice crystal diameter compared to the untreatedk-carrageenan Hydrolysis times shorter than 20 min do not exhibit a statistically significant influence on the mean ice crystal diameter during thefirst 3 h of storage time Here, it is important to mention that the values of mean ice crystal diameters after 3 h storage time are not the initial ice crystal sizes Due to experimental setup and sample preparation, it was not possible to take pictures before 3 h storage time But even if we cannot completely eliminate the option that k-carrageenan affects ice nucleation, we assume that the initial ice crystal diameters are the same for all solutions (see section2.4.3)

After 96 h of storage time and, therefore, a longer time period for recrystallization processes, the influence of hydrolysis on the IRI activity ofk-carrageenan becomes more clear It is apparent that the mean ice crystal diameters increase with increasing hydrolysis time However, the differences in mean ice crystal diameters of the hydrolyzed samples compared to the untreated sample are only statistically significant after hydrolysis times of 20 and 60 min The pH value of all hydrolyzedk-carrageenan sucrose solutions was about 4 Because the pH was lower than the pH of the un-treatedk-carrageenan sucrose solution (pH 6), it might be possible that not all TFA was evaporated during freeze-drying However, as all hydrolyzedk-carrageenan solutions have the same pH, the re-sidual amounts of TFA in the different hydrolyzedk-carrageenan samples should be similar In addition, as there is no significant difference in IRI activity of untreated (pH 6) and 5 min hydrolyzed

k-carrageenan (pH 4), an inﬂuence of non-evaporated residual TFA

on IRI activity can be excluded

As we used the same mass concentration of k-carrageenan samples (1 mg mL1) in this recrystallization experiment, the molar concentration ofk-carrageenan is signiﬁcantly higher in solutions with hydrolyzed k-carrageenan due to the smaller molecular weight An inﬂuence on the ice content can be neglected as the molar concentration of the hydrolyzedk-carrageenan is too low to

inﬂuence the equilibrium phase volume of ice Therefore, we can conclude thatk-carrageenan molecules that have a lower molec-ular weight due to TFA hydrolysis exhibit a reduced IRI activity However, the hydrolyzedk-carrageenan samples still show IRI ac-tivity, because the mean ice crystal diameters of all hydrolyzed samples are signiﬁcantly smaller than the mean ice crystal di-ameters in pure sucrose solution

The question arises why the IRI activity of smallk-carrageenan molecules is reduced Assuming that IRI activity ofk-carrageenan originates from an interaction with the ice crystal surface, a conformational change of thek-carrageenan molecules could be a reason for the decreased IRI activity For example,Abad et al (2008) did not observe a conformational change from coil to helix ofk -carrageenan molecules with a weight average molecular weight

Mwof 34 kDa, which is in a similar range as the molecular weight of the used hydrolyzedk-carrageenan samples (seeFig 2) Therefore, the coil-helix transition was investigated InFig 4the temperature sweep analysis of the untreated k-carrageenan extract and the hydrolyzed samples (5 min and 60 min) dissolved in 49% sucrose solution are shown It is apparent that there is a coil-helix transition

in solution with untreatedk-carrageenan extract at a temperature

of about 25C In addition, a small thermal hysteresis and thus aggregation is detectable Aggregation is probably due to the presence of cations in thek-carrageenan extract (Leiter et al., 2016a,

2017) In contrast, no coil-helix transition and aggregation is detectable in both hydrolyzedk-carrageenan solutions These re-sults are in agreement with those obtained byAbad et al (2008) Because there is no signiﬁcant difference in IRI activity of untreated and 5 min hydrolyzedk-carrageenan (seeFig 3), it seems that coil-helix transition is not a necessary condition for IRI activity How-ever, we cannot completely exclude a coil-helix transition asRochas

Fig 2 Size-exclusion chromatograms of untreatedk-carrageenan extract and

freeze-driedk-carrageenan samples hydrolyzed with 0.1 M triﬂuoroacetic acid for different

times The signal intensities are arbitrary, but the same scale is used for the refractive

index signals of all curves.

Fig 3 Inﬂuence of different hydrolysis times with 0.1 M triﬂuoroacetic acid on ice

recrystallization inhibition activity ofk-carrageenan dissolved in a sucrose solution

(w suc ¼ 49% (w/w), ck-carr ¼ 1 mg mL 1 ) Bars with the same small letter at the same

storage time are not signiﬁcantly different (P > 0.05).

Trang 6

et al (1990)detected a coil-helix transition even fork-carrageenan

molecules with a Mwof about 6 kDa Possibly, the transition

tem-perature is lower than 0 C as coil-helix transition temperature

decreases with decreasing molecular weight (Meunier et al., 2001)

Furthermore, measurements of G’ in this low range might be

insufﬁciently sensitive to detect the coil-helix transition Therefore,

further investigations on the relation between k-carrageenan

conformation and IRI activity are necessary

3.3 Molecular weight distribution and IRI activity ofk-carrageenan

after dialysis

Beside the acid hydrolysis with 0.1 M TFA, the molecular weight

ofk-carrageenan was reduced by using dialysis.Hoffmann et al

(1996)showed that molecular weight decreases with increasing

dialysis time of a 2 mg mL1k-carrageenan solution against Milli-Q

water and subsequent freeze-drying

It can be seen from the results shown inFig 5that 36 h dialysis

of the untreatedk-carrageenan extract against demineralized

wa-ter and subsequent freeze-drying leads to a signiﬁcant reduction of

the molecular weight The molecular weight of the main peak Mpis

shifted from 1420 kDa to about 23.1 kDa In contrast, the molecular

weight reduction is signiﬁcantly lower, when k-carrageenan is

additionally dialyzed against a KCl solution before freeze-drying

However, this procedure also yields a lower molecular weight

compared to the molecular weight of the untreatedk-carrageenan

extract The main peak of thek-carrageenan additionally dialyzed

against a KCl solution is shifted from 1420 kDa to 219 kDa while a

small molecule fraction remains in the range of 1420 kDa In

addition, the molecular weight distribution is much broader if

compared to thek-carrageenan sample dialyzed against

deminer-alized water

Hoffmann et al (1996)provided a possible explanation for the

signiﬁcantly lower molecular weight of the dialyzedk-carrageenan

in comparison to the dialyzedk-carrageenan with the addition of

Kþcations The authors showed that dialysis against demineralized

water leads to a conversion of thek-carrageenan molecule into the

acid form causing a pH drop In our case, the pH of thek

-carra-geenan solution in the dialysis tubing dropped from about 6 to 3

during 36 h of dialysis According toHoffmann et al (1996)this pH decrease already induces depolymerization of k-carrageenan In addition, they assumed that the concentration of the sample during freeze-drying will further lower the pH at a local level which causes

an increased depolymerization They further supposed that if an excess of sulfate-bound cations, such as potassium, is present during freeze-drying, there is no acid form of thek-carrageenan molecule and thus additional depolymerization does not occur This assumption ofHoffmann et al (1996)is in agreement with our results that both the molecular weight and the IRI activity are un-affected when the untreated k-carrageenan was dissolved in demineralized water and freeze-dried with its initial cations (data not shown) Therefore, the molecular weight reduction of thek -carrageenan dialyzed against demineralized water and KCl is most likely derived from an autohydrolysis during the dialysis, whereas additional molecular weight reduction of thek-carrageenan dia-lyzed against demineralized water is mostly derived from an increased autohydrolysis during freeze-drying of the acidk -carra-geenan form

InFig 6, the mean ice crystal diameters in frozen sucrose so-lutions with the different dialyzedk-carrageenan samples after 3 h and 96 h of storage time are depicted Larger mean ice crystal di-ameters and thus a reduced IRI activity were observed for samples with a lower molecular weight The small reduction of the molec-ular weight of the k-carrageenan dialyzed against demineralized water and KCl leads to a small increase in the mean ice crystal diameter after 96 h compared to the untreated k-carrageenan However, this difference is not statistically signiﬁcant In contrast, the mean ice crystal diameters in thek-carrageenan solution dia-lyzed against demineralized water are signiﬁcantly larger than in the solution containing untreatedk-carrageenan extract

To further investigate whether smallerk-carrageenan molecules exhibit a reduced IRI activity, we hydrolyzed the dialyzedk -carra-geenan with 0.1 M TFA for 60 min to further reduce the molecular weight For this sample, the reduction of molecular weight leads to

a total loss of IRI activity as there is no signiﬁcant difference be-tween the mean ice crystal diameters in a pure sucrose solution and

in a solution containing the dialyzed and subsequently hydrolyzed

k-carrageenan sample

Fig 4 Temperature sweep analysis (2C min1) of untreatedk-carrageenan extract

(c k -carr ¼ 1 mg mL 1 ) andk-carrageenan hydrolyzed with 0.1 M triﬂuoroacetic acid for

5 or 60 min in 49% (w/w) sucrose solution To distinguish between the data points of

hydrolyzedk-carrageenan samples, only every second data point of the 60 min

hy-drolyzed sample is shown.

Fig 5 Size-exclusion chromatograms of untreatedk-carrageenan extract,k -carra-geenan dialyzed against demineralized water, andk-carrageenan dialyzed against demineralized water and subsequently dialyzed against 2.5 mM KCl before freeze-drying The signal intensities are arbitrary, but the same scale is used for the refrac-tive index signals of all curves.

Trang 7

Thus, these results are in good agreement with the results of the

0.1 M TFA hydrolysis Both experiments show that the reduction of

molecular weight leads to a decrease in IRI activity

This relationship between molecular weight and IRI activity was

also found for polyvinyl alcohol (PVA) and for the ice binding

protein AFGP (Budke et al., 2014; Congdon et al., 2013; Inada and Lu,

2003) Both PVA and AFGP exhibit strong IRI activity and bind to the

ice crystal surface.Budke et al (2014)postulated that,ﬁrstly, an

increased molecular weight and thus an increased molecular size

may result in a larger surface area covered per molecule and,

sec-ondly, larger molecules exhibit more potential moieties for the

adsorption to the ice, which may lead to a faster and/or stronger

adsorption Assuming that IRI activity ofk-carrageenan also

origi-nates from an interaction with the ice crystal surface, this

expla-nation is also conceivable for the inﬂuence of the molecular weight

of k-carrageenan on its IRI activity However, these ﬁndings are

contrary to the study ofKaminska-Dworznicka et al (2015)who

showed that k-carrageenans hydrolyzed by sulfuric and

hydro-chloric acid have an increased IRI activity Possibly, hydrolysis with

sulfuric and hydrochloric acid results in a different molecular

structure of degradedk-carrageenan with an increased IRI activity

To evaluate this hypothesis, the effect ofk-carrageenan hydrolysis

with HCl on the IRI activity was also studied However, Naþcations

from NaOH, which was used to neutralize the acid solution, can

lead to aggregation It was already demonstrated that aggregation

of k-carrageenan molecules decreases IRI activity (Leiter et al.,

2016a) Therefore, aggregation was additionally analyzed by

rheo-logical temperature sweep measurements

3.4 Molecular weight distribution and IRI activity ofk-carrageenan

after acid hydrolysis with 0.1 M HCl

The hydrolysis with 0.1 M HCl leads to a signiﬁcant reduction of

molecular weight (Fig 7) The main peak is shifted from 1420 kDa

to 262 kDa, and the second peak at a retention time of 370 min

strongly increases This strong increase is mainly due to the NaCl

content in the freeze-dried sample, which originates from the HCl

used for hydrolysis and the NaOH used for neutralization In our

previous studies, we showed that the presence of Naþcations

re-duces IRI activity of k-carrageenan due to aggregation of the

molecules (Leiter et al., 2016a, 2017) Therefore, the same ion concentration has to be present in the hydrolyzedk-carrageenan and the untreatedk-carrageenan to allow a comparison of the IRI activity For this reason, we made a reference sample by adding the same NaCl amount that is present in the hydrolyzedk-carrageenan sample to the untreatedk-carrageenan extract and freeze-dried this sample without hydrolysis In the following, this reference sample is termed sample A and the hydrolyzed k-carrageenan sample is termed sample B The ion content in sample A and sample

B is equal and the inﬂuence of ions on IRI activity is the same in both samples Thereby, an investigation on the inﬂuence of the molecular weight on IRI activity is possible

To investigate the influence of hydrolyzedk-carrageenan on IRI activity, freeze-dried samples A and B were dissolved in 49% su-crose solutions to obtain the same concentration (1 mg mL1) as in the previous experiments However, due to the high amount of ions, thefinalk-carrageenan concentration in these sucrose solu-tions is only 0.146 mg mL1 Therefore, sucrose solutions with a concentration of 6.844 mg mL1of sample A or B leading to afinal

k-carrageenan concentration of 1 mg mL1were also investigated Thefinal salt and k-carrageenan concentrations in the different sucrose solutions with sample A or B are listed inTable 1 For solutions with a lower sample concentration (1 mg mL1), the hydrolyzed k-carrageenan sample (sample B) shows slightly higher mean ice crystal diameters thank-carrageenan with NaCl (sample A) after 3 h and 96 h (Fig 8a) However, the difference is only significant at 3 h of storage time For higher sample concen-trations (6.844 mg mL1), no significant differences between the mean ice crystal diameters were observed for samples A and B

Fig 6 Mean ice crystal diameters in frozen sucrose solutions with different

depoly-merizedk-carrageenan samples (w suc ¼ 49% (w/w), ck-carr ¼ 1 mg mL 1 ) after 3 h and

96 h of storage time at 12 C Molecular weight was reduced by dialyzingk

-carra-geenan against demineralized water and by hydrolyzing the dialyzedk-carrageenan

with 0.1 M triﬂuoroacetic acid Bars with the same small letter at the same storage time

are not signiﬁcantly different (P > 0.05).

Fig 7 Size-exclusion chromatograms of untreated k-carrageenan extract and k -carrageenan that was hydrolyzed with 0.1 M HCl for 30 min, neutralized with NaOH, and freeze-dried The signal intensities are arbitrary, but the same scale is used for the refractive index signals of all curves.

Table 1 NaCl andk-carrageenan concentrations for samples A and B dissolved in 49% sucrose solution.

Concentration of sample A or B [mg mL1]

Final mass concentration

ofk-carrageenan [mg mL1]

Final mass concentration

of NaCl [mg mL1]

Final molar concentration

of NaCl [mmol L1]

Trang 8

(Fig 8b) Thus, hydrolysis ofk-carrageenan with 0.1 M HCl at 60C

for 30 min has no signiﬁcant effect on its IRI activity

However, the increase in concentration of sample A and B from

1 mg mL1to 6.844 mg mL1and thus an increase in thek

-carra-geenan concentration from 0.146 mg mL1 to 1 mg mL1(see

Table 1) leads to a signiﬁcant increase in mean ice crystal diameters

The mean ice crystal diameters in solutions with a sample

con-centration of 6.844 mg mL1 are about 40mm (Fig 8b) and are

similar to the mean diameter of ice crystals in pure sucrose solution

which is about 46mm (seeFigs 3 and 6) Thus, there is hardly any

IRI activity left in the solutions with large sample concentrations of

sample A or B, independently whether or not k-carrageenan is

hydrolyzed This result is rather surprising because increasingk

-carrageenan concentrations in sucrose solutions usually results in

increasing IRI activities (Gaukel et al., 2014) This was also observed

for the untreatedk-carrageenan solution, which exhibits a smaller

mean ice crystal diameter at a higher concentration For example,

the mean ice crystal diameter after 96 h storage time decreases

from 16mm (Fig 8a) to 10mm (Fig 8b) when the concentration of

the untreated k-carrageenan increases from 0.146 mg mL1 to

1 mg mL1in pure sucrose solutions Therefore, it is likely that the

large salt concentration of 100 mM is responsible for the decreased

IRI activity due to aggregation ofk-carrageenan molecules

To prove this hypothesis, a temperature sweep analysis was

performed (Fig 9) It is evident that increasing the concentration of

sample A and B from 1 mg mL1to 6.844 mg mL1leads to an

increase of aggregation In a sucrose solution with a low

concen-tration of sample A (1 mg mL1) there is a small thermal hysteresis

and thus aggregation is detectable In this solution the NaCl

con-centration is 14.6 mM (see Table 1) In addition, the coil-helix

transition is at about 10 C By increasing the concentration of

sample A to 6.844 mg mL1and thus the NaCl concentration to

100 mM, the thermal hysteresis is signiﬁcantly higher

Conse-quently, the degree of aggregation increases Additionally, the

coil-helix transition is shifted from 10C to 30C This is in agreement

with previous studies showing that coil-helix transition is shifted to

higher temperature with increasing cation concentration (Doyle

et al., 2012; Rochas and Rinaudo, 1980) In a sucrose solution

con-taining the hydrolyzed sample B at low concentration (1 mg mL1)

neither aggregation nor coil-helix transition are detectable in

accordance with the results of the TFA hydrolysis shown inFig 4

However, by increasing the concentration of the hydrolyzed sample

B to 6.844 mg mL1a large thermal hysteresis is detectable The lower coil-helix transition temperature of 20C compared to the transition temperature of 30C of sample A is in agreement with the results of Meunier et al (2001)who showed that coil-helix transition temperature decreases with decreasing molecular weight Finally, it is apparent that in both sucrose solutions with higher sample concentrations and thus higher NaCl concentrations the degree of aggregation is increased Thus, these results conﬁrm the hypothesis that the reduced IRI activity at higher sample con-centrations is due to a higher degree of aggregation

3.5 Inﬂuence of NaCl concentration on IRI activity Surprisingly, the IRI activity of sample A and B at low concen-trations is signiﬁcantly increased relative to untreated k -carra-geenan (Fig 8a) For example, after 96 h the mean ice crystal diameters in sucrose solutions with low concentrated sample A or B

Fig 8 Mean ice crystal diameters in frozen sucrose solutions (w suc ¼ 49% (w/w)) with untreatedk-carrageenan extract, untreatedk-carrageenan with NaCl (sample A), ork -carrageenan ﬁrst hydrolyzed with 0.1 M HCl and then neutralized with NaOH (sample B) after 3 h and 96 h of storage time at 12 C The same NaCl contents are present in sample

A and B (a) c k -carr ¼ 0.146 mg mL 1 , c sample A or B ¼ 1 mg mL 1 (b) c k -carr ¼ 1 mg mL 1 , c sample A or B ¼ 6.844 mg mL 1 Bars with the same small letter at the same storage time are not signiﬁcantly different (P > 0.05).

Fig 9 Temperature sweep analysis (2C min1) of freeze-dried sample of untreatedk -carrageenan extract with NaCl (sample A) andk-carrageenan hydrolyzed with 0.1 M HCl for 30 min and subsequent neutralization with NaOH (sample B) dissolved in 49% (w/w) sucrose solution (c sample ¼ 1 mg mL -1 or 6.844 mg mL1) Both freeze-dried samples have the same mass ratio of NaCl tok-carrageenan.

Trang 9

are signiﬁcantly smaller than ice crystal diameters in sucrose

so-lution with the same amount of untreated k-carrageenan

(0.146 mg mL1) without additional salt The mean ice crystal

di-ameters in these solutions with sample A and B are nearly of the

same size as ice crystal diameters in sucrose solution with a higher

k-carrageenan concentration of 1 mg mL1(Fig 8b) Possibly, the

addition of a small amount of NaCl, which induces either no or only

a low degree of aggregation, leads to an increased IRI activity ofk

-carrageenan For example, we showed in a previous study that the

addition of NaCl up to a limiting value between 20 and 30 mM to a

solution with the ice binding protein AFP III leads also to an

increased IRI activity (Leiter et al., 2016b) To further verify the

assumption that a small amount of salt increases the IRI activity of

k-carrageenan, we added the same small amount of NaCl (14.6 mM)

to the sucrose solution with the higherk-carrageenan

concentra-tion of 1 mg mL1

InFig 10, the inﬂuence of the molar NaCl concentration on the

mean ice crystal diameter ink-carrageenan sucrose solution after

96 h storage time is shown The mean ice crystal diameter in thek

-carrageenan sucrose solution with a ﬁnal NaCl concentration of

14.6 mM is signiﬁcantly smaller than in purek-carrageenan sucrose

solution (P< 0.05) Whereas at higher NaCl concentrations the IRI

activity is reduced due to a higher degree of aggregation as

ex-pected However, the addition of a very small NaCl amount

(0.3 mM) does not inﬂuence IRI activity, which is in good

agree-ment with the observation that the addition of 0.3 mM KCl also

does not inﬂuence the IRI activity (Leiter et al., 2016a) Thus, there is

probably only a small concentration range where IRI activity is

increased However, the reason for this remains unclear and further

investigations are necessary

The results ofFigs 8 and 10show that the salt concentration can

inﬂuence the IRI activity in different ways A high salt concentration

leads to aggregation and reduces IRI activity whereas a low salt

concentration can improve the IRI activity Hence, the addition of

salt (due to the utilization of a buffer system or the necessity of

neutralization) should ideally be avoided for investigations on the

inﬂuence of the molecular weight or the molecular structure ofk

-carrageenan on its IRI activity However, if this is not possible,

recrystallization experiments should be performed at least at the

same salt concentration In addition, as IRI activity is lost at a high

degree of aggregation, salt concentrations should be selected

carefully so that no or only a low degree of aggregation occurs

Possibly, this explains the contrary results of Kaminska-Dworznicka et al (2015) In this study, the IRI activity of a neutralized untreatedk-carrageenan sample was compared with a neutralized hydrolyzedk-carrageenan sample in sucrose solution However, in their study different concentrations of the untreated and hydrolyzed samples and thus different salt concentrations were used for recrystallization experiments (0.01% of the neutral-ized untreatedk-carrageenan sample and 0.005% of the neutralized hydrolyzedk-carrageenan sample) Thus, the higher IRI activity was possibly derived from a lower degree of aggregation in the solution with hydrolyzedk-carrageenan due to the lower salt concentration and not from a hydrolysis of thek-carrageenan molecules

4 Conclusion

In this study, the inﬂuence of the molecular weight ofk -carra-geenan on its IRI activity was investigated The molecular weight was reduced by acid hydrolysis or by dialysis against demineralized water Dialysis and subsequent freeze-drying of the acid form ofk -carrageenan led to a signiﬁcant reduction of the molecular weight

ofk-carrageenan whereas higher molecular weights were observed when sulfate-bound cations were present during freeze-drying It was demonstrated that IRI activity ofk-carrageenan decreases with decreasing molecular weight Assuming that IRI activity of k -carrageenan originates from an interaction with the ice crystal surface, we suppose that larger molecules do probably exhibit more potential moieties for an interaction with the ice crystal surface, which may lead to a faster and/or stronger adsorption Further-more, we showed that the addition of NaCl can inﬂuence the IRI activity in different ways A high salt concentration leads to an aggregation of thek-carrageenan and reduces IRI activity whereas a small salt concentration can improve the IRI activity However, the reason for this increase in IRI activity is unclear

As salt is present in almost all food products, future research should focus on possibilities to avoid aggregation in the presence of salt In addition, more research is necessary to provide a better understanding of the IRI mechanism of k-carrageenan For this purpose, more detailed studies on the inﬂuence of ions on IRI ac-tivity are required

Acknowledgement The authors would like to thank Welding GmbH& Co KG and Eurogum A/S for providing thek-carrageenan extract

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