Effect of Pectins on Water Crystallization Pattern and Integrity of Cells During Freezing

Abstract

The ability of various pectin polysaccharides to modify the morphological structure of ice during the phase transitions from water to ice was studied. Pectins were isolated from Sosnowsky's hogweed Heracleum sosnowskyi Manden (heracleuman—N6HS), tansy Tanacetum vulgare L. (tanacetan—N7TVF), and Rauwolfia serpentina Benth callus (rauwolfian—N8RS). Pectins were isolated by multistep extraction. The effect of pectins was assessed using osmometry, thermographic analysis, and cryomicroscopy. A concentrate of leukocytes was used as the sample for the subsequent freezing step. The condition of the leukocyte membrane, and lysosomal and phagocytic activity after a freezing–warming process were assessed. Osmotic concentrations of the pectin polysaccharide solutions were found to be very low. The 0.4 wt % N7TVF solution had the highest osmotic concentration as well as freezing point; however, the duration of its crystallization plateau was lower than that of the 0.4 wt % and 0.2 wt % N6HS solutions. All studied polysaccharide solutions demonstrated a high linear rate of ice crystal growth. There were statistically significant differences between the melting rates for the 0.2% solutions of the pectins, N6HS and N7TVF, N6HS and N8RS, as well as between concentrations for the pectin N7TVF and between concentrations for the pectin N8RS. The data on the integrity of cells that are frozen in a medium containing polysaccharides may indicate a cryoprotective effect of the N7TVF and N8RS pectins, that is, tanacetan from tansy and rauwolfian from rauwolfia. The most effective modifier among the substances, which were studied by us, was the N7TVF pectin polysaccharide (tanacetan from tansy).

Introduction

The response of any life form to lowering environmental temperatures is to launch a survival program predetermined by genes. One such specific defense mechanism is the induction of extracellular crystallization at −5°C to −10°C by the substances known as nucleating agents (NA). Biological NA are widespread in nature. Currently they are widely studied in bacteria,^1–3 animals,^4–9 and fungi.^10,11

The data on plant NA are less ample. It is known that the leaves of frost-resistant fall rye Secale cereale contain a compound, glycophospholipoglycoprotein, which triggers nucleation at a threshold temperature of −7°C; however, the frequency of its occurrence is 1–105 mesophyll cells of the leaves.¹² NA were identified in peach shoots, Prunus sp., first emerging in young shoots in late summer and reaching its peak activity by late August. Their characteristic traits are the nucleation temperature of −2°C and nonprotein origin.¹³ Highly active NA were identified in the extracellular fluid of white fir Abies concolor and Chinese juniper Juniperus chinensis,¹⁴ in common sea-buckthorn Hippophae rhamnoides,¹⁵ in Citrus sinensis fruits,¹⁶ and in different varieties of grapes.¹⁷ In blueberry, nucleators are initiated periodically, following seasonal changes.¹⁸

The study of published articles on plant NA shows that it was difficult to extract these substances from plant tissues in the 1980s and 1990s, which is probably the reason behind the paucity of further research on the topic.¹⁹ Today new biotechnological methods exist that make extracting plant components possible and evaluating their chemical structure, so that the previous studies can now be resumed.

Another way of protecting against the adverse effect of cold temperatures is the synthesis of such substances as cryoprotectants, which serve to mitigate cold-induced damages in species subject to low temperatures. The nature and cryoprotectant action of a component in viscous mucus-like substance of the blooms of Lobelia telekii, an Afro–Alpine cold-hardened plant exposed to frosts at nighttime, has been widely debated. Some authors are of the opinion that the crystallization of this fluid is induced in the presence of a large polysaccharide having a highly specific activity even if its content is low,²⁰ whereas other authors suppose that this viscous substance is an antifreeze agent conducive to the cooling of fluids in plant tissues.²¹ Other findings support the hypothesis in that this substance is neither an antifreeze nor a NA, but a pectin protecting the bloom fluid storage from evaporation and drying up.²² It is also known that the content of various pectins (the degree of methyl esterification, the branching of the side chains) in the cell wall of different pea varieties affects their frost resistance.^23,24 It is assumed that pectins with branched chains are more capable of gel formation and can protect cells from dehydration.^25,26 Apart from the water binding, the pectins are able to protect the cell against cytotoxicity generated by H₂O₂ through the reduction of intracellular reactive oxygen species levels.²⁷

Previously, we demonstrated that a pectin polysaccharide added to a cryopreservative solution serves to increase the percentage of fully functional cells after thawing.^28–32

This article aims at defining a role of pectin polysaccharides for water crystallization processes during freezing.

It should be noted that biological functions and physiological activity of plant polysaccharides are determined by their individual structures.^33–35 More specifically, as a part of the comprehensive study of chemical composition and physiological activity of pectin substances in plants of the North of European Russia, an appropriate procedure of polysaccharide extraction from plants was developed, their structures were defined, and their immunological activity was measured.^35–37

Pectin substances belong to a larger group of glycanogalacturonans and include protopectin, pectin polysaccharides (homogalacturonan, rhamnogalacturonans I and II [RG-I and RG-II], xylogalacturonan and apiogalacturonan) and associated arabinans, galactans, and arabinogalactans. They form a part of cell walls of all virtually higher terrestrial and water plants to perform a number of important biological functions such as mediating ionic transport and water economy, enhancing plant resistance to drought and frost, and acting as protectants in plant–phytopathogen relationship.^35,38 In this regard, plants which grow under humid conditions and at low temperatures are especially noteworthy, as some of their components may be considered potential organic cryoprotectants to serve as a foundation for the development of new cryopreservation techniques.

Materials and Methods

We used the following pectin polysaccharides extracted and characterized by the Department of Immunology and Biotechnology, Institute of Physiology, Ural Division Research Center of Russian Academy of Sciences: N6HS—heracleuman from Sosnowsky's hogweed Heracleum sosnowskyi Manden, N7TVF—tanacetan from tansy Tanacetum vulgare L., N8RS—rauwolfian from Rauwolfia serpentina Benth callus.

The following methods were employed.

Osmometry

Osmotic concentrations were evaluated using the OMKA-C-01 osmometer (Medlabortekhnika). The solution under study was placed into the measuring cell of the osmometer, then a thermosensitive gauge was plunged into the sample, and the well was placed into a cooling system. The tolerance for 0–400 mOsm/L range was ±2.0.

Thermographic analysis

One milliliter of plant polysaccharide solution was introduced to 2 mL Nunc cryovials. Cryovials containing samples were placed into the freezing chamber and cooled at the rate of 1°C/min. The temperature was measured directly in the sample using OVEN TRM 200 multipurpose two-channel measuring device.

Cryomicroscopy

Cryomicroscopy was employed to study the specific morphology of the crystalline structure forming during the phase transitions in plant polysaccharide solutions and to evaluate the ice crystal melting rate.

The following experimental groups were studied: a solution of pectin N6HS at a concentration of 0.2 wt %, a solution of pectin N6HS at a concentration of 0.4 wt %, a solution of pectin N7TVF at a concentration of 0.2 wt %, a solution of pectin N7TVF at a concentration of 0.4 wt %, a solution of pectin N8RS at a concentration of 0.2 wt %, and a solution of pectin N8RS at a concentration of 0.4 wt %. In the study of cell freezing, control rate freezing was carried out with a solution of glycerol at a concentration of 7%. Polysaccharide solution in different (0.2 wt %, 0.4 wt %) concentrations were prepared using distilled water as solvent. The concentrations above were selected according to the earlier data on pectin polysaccharides as components of cryoprotectant media.^28–32

The samples were transferred by a pipette on a slide to the cryochamber. Small AgJ crystals were introduced to the droplets to eliminate supercooling and visualize the field of vision. The droplet was covered with a cover glass to avoid drying and to ensure that the layer is sufficiently thin to produce a clear image when viewed against the light. The samples were cooled at the rate of 1°C/min to facilitate completion of the visible changes in morphology of the crystalline structure (−15°C to −20°C) and then warmed at the rate of 1°C/min. The temperature values during cooling were controlled from the operating chamber of the cryotable. The kinetic properties of crystallization and melting were recorded using the DCM-300 camera lens in the photo and frame-by-frame video recording modes (1 frame/5 s or 1 frame/3 s, each subsequent frame matching the decrease or increase of the temperature in the operating chamber by 0.08°C or 0.05°C, respectively).

Ice crystal sizes at the melting stage were evaluated using the Aim Image Ex computer application.

An additional series of tests was conducted to determine the effect of the pectin polysaccharides studied for human blood cell cryopreservation.

Leukocytes were extracted from whole volunteer donor blood (aged 39.4 ± 12.2) by cytapheresis (2500 rpm, 5 minutes with cooling; Sorvall). The cells were mixed (1:1) before freezing with the cryoprotectant solution in Kompoplast 300 flexible PVC container and exposed for 20 minutes at normal ambient temperature. The cryopreservative components were: 7% glycerol (nontoxic concentration) and one of the above pectin polysaccharides, concentration 0.2 wt %, and Trilon B (0.1%), which was used as an anticlotting agent.

The mixture was frozen for 15 minutes in an alcohol (98% ethyl alcohol) bath cooled to −28°C. The rate of cooling from room temperature to the eutectic point (−2°°C) was 10°C/min, then down to −20°C, and the rate of cooling was 2–3°C/min. The samples were stored for 24 hours at the specified temperature in an electric freezer in the air environment. The sample was warmed for 40 seconds in a 20-L water bath (+38°C), while the container was actively tilted. The following factors were assessed using light microscopy (H550S; Nikon): (1) the total number of leukocytes in Goryaev chamber, (2) the cryoresistance of different cell populations in smears immersed in May-Grünwald and Romanowsky stains, and (3) the integrity of leukocyte cell membrane in samples immersed in 1.0% Eosin Supravital stain solution. The diffused pink staining of cytoplasm was considered an indicator of cell membrane damage, whereas viable cells with an undamaged membrane had a light green color.

Neutrophil granule protein activity that is responsible for a killer effect was evaluated by lysosomal cation test using the procedure proposed by Slavinsky and Nikitina.³⁹ This procedure is also useful for evaluation of the depth of low-temperature neutrophil damage as lysosome membranes are highly sensitive to freezing and susceptible to losing their barrier properties in this way, which, in turn, leads to hydrolase leakage to cytoplasm and brings about secondary, or “latent,” cell damage. Neutrophil phagocytic activity was evaluated relying on a modified method proposed by Potapova et al.⁴⁰ and using 0.08 μm inert latex particles (Sigma-Aldrich). Only the percentage of phagocytic cells was determined. The number of captured particles (phagocytic index) for all samples before freezing matched Grade II (11–30 particles) and Grade I after warming (10 particles or less).

Statistical data manipulation involved calculation of the arithmetic mean value ± standard deviation (M ± δ). The Wilcoxon signed-rank test was applied to determine statistical significance of differences between the groups using BIOSTAT, a computer application for medical and biological statistics.⁴¹

Results

Osmotic concentrations of the pectin polysaccharide solution studies were found to be very low (osmotic concentration of the distilled water being 23.3 mOsm/L). The 0.4 wt % N7TVF solution had the highest osmotic concentration as well as freezing point; however, the duration of its plateau of crystallization is lower than that of the 0.4 wt % and 0.2 wt % N6HS solutions (Table 1).

Table 1.

Osmotic and Hydrographic Characteristics of Aqueous Solutions of Plant Pectins

No.	Pectin's concentration in solution (weight/volume)	Osmolarity, mOsm/L	Freezing point, °C	Plateau's temperature, °C	ΔT, °C	Plateau's time, minutes
1	0.4% N6HS	35.0	−4.0	0	4.0	13.48
2	0.2% N6HS	34.0	−3.739	−0.12 to −0.19	3.618	9.23
3	0.4% N7TVF	42.0	−7.230	−0.072	7.158	7.21
4	0.2% N7TVF	24.0	−5.870	−0.057	5.813	5.26
5	0.4% N8RS	39.0	−7.053	−0.148	6.905	4.25
6	0.2% N8RS	31.0	−5.342	−0.107	5.235	4.03

Analysis of crystallization and melting process kinetics in N6HS pectin solutions

Small AgJ crystals in the reference sample are positioned chaotically in the field of vision. AgJ crystals suspended in the polysaccharide solution move so as to change their density in the field of vision. At a temperature of −1.9°C, the phase transition is accompanied by the formation of unidirectional parallel linear channels, into which the AgJ crystals were pushed. The movement of AgJ particles inside these channels was indicative of the fact that visible crystallization processes in the sample at the range of −2.62°C to −4.22°C continued. No visible changes were observed in the morphology of the sample during further cooling. It is only at the temperatures below −10°C that the local areas containing concentrated N6HS pectin polysaccharide solution were identified; these areas having formed as a result of water freezing out in the channels.

As for warming, it is the increase of the above areas owing to water supply that signaled the commencement of the slow melting phase. The intense melting phase was registered at the range of temperatures of −4.04°C to −3.48°C. Complete melting of the sample in the field of vision was observed at −3.4°C. It should be noted that the linear pattern of AgJ crystals persisted as an image of the morphology of the crystalline structure. Such a crystallization pattern of 0.2% N6HS solutions was observed in all iterations of the tests (n = 5).

The movement of AgJ crystals in 0.4% N6HS solution was very limited. The original pattern in the solution remains virtually the same during the cooling–warming cycle. The solution freezing point was recorded at −1.56°C with the associated formation of a branched channel network. During warming, the clear image of the narrow channel network persisted up until the temperature of −4°C. As the temperatures increased, the channels expanded continuously, and the sample completed its melting.

Analysis of crystallization and melting process kinetics in N7TVF pectin solutions

The phase transitions for the 0.2 wt % N7TVF pectin solution was observed at −1.76°C and was associated with the linear growth of the crystalline structure, which had spread in the field of vision almost rapidly, and with the formation of linear liquid fraction channels, just as with the 0.2% N6HS pectin solution. During further cooling, the freezing out of water in the liquid fraction was associated with continuous narrowing of the channels and the expected increase of polysaccharide concentrations inside these channels. Visible melting in the sample was registered at around −10°C starting from localized and later expanding areas. The intense melting area (−3.56°C to −3.32°C) is characterized by rapid channel expansion until the complete disappearance of ice crystals from the field of vision.

The specific feature of the morphological structure, which forms during the phase transitions in the 0.4 wt % N7TVF pectin solution at −1.88°C, is the initial formation of a fairly dense channel network, some of which are strictly linear, whereas the others are multidirectional and deformed. A wide channel in this structure, clearly identifiable by the separated AgJ crystals, was the boundary between these areas. There is a strong possibility that in this case, there were several spots from where the crystals had sprung, or that the initial linear frontline lost its stability due to the concentration gradient, which had emerged during the phase transitions and led to the irregular growth of crystals. The possibility of the effect of the pectin-specific macromolecular structure on ice crystals' morphology cannot be ruled out.

Ice crystal melting kinetics of the 0.4 wt % N7TVF pectin solution showed no principal differences from that of the 0.2 wt % N7TVF pectin solution. Visual evidence of melting was clearly identified in the channel which separated the crystalline structures of the sample as early as at −10°C. As the temperature increased, the channel expanded fairly slowly up until −3.96°C. It was followed by the intense melting area at the range of temperatures of −3.56°C to −3.24°C until the complete disappearance of ice crystals from the field of vision.

Analysis of crystallization and melting process kinetics in N8RS pectin solutions

The phase transitions for the 0.2 wt % N8RS pectin solution was observed in the field of vision at −2°C and was associated with the formation of a one-way, slightly deformed linear structure of narrow channels, all of which froze during further cooling to −20°C. The only evidence of their presence is the spot positioning of the AgJ crystals separated into these channels by the growing frontline of ice crystals. During warming, the channel lines become barely visible at −10°C and more clearly visible only when the temperature reaches −3.85°C. Intense melting is in the range of temperatures from −3.2 to −3.1°C, until the complete disappearance of ice crystals from the field of vision at −3.05°C.

The phase transition for the 0.4 wt % N8RS pectin solution is registered starting from −2°C. The morphology of the crystalline structure, which formed in the 0.4 wt % N8RS pectin solution, is very similar to that of the initial crystalline structure in the 0.4 wt % N7TVF pectin solution: two-way crystal growth is also identified by the presence of a boundary channel. However, the channels are virtually parallel and the liquid nondeformed in this sample. As the temperature drops, liquid fraction in the channels freezes out, so that some of these disappear completely. Only the larger channels containing a large portion of separated AgJ crystals are clearly visualized. The melting process is associated with visible channel expansion commencing at −10°C. Gas bubbles are released in the intense melting area, which then gradually dissolve as the temperature rises. Ice crystals are completely melted in the field of vision at −3.08°C.

As the cryomicroscopy tests show, the linear growth rate of ice crystals for all studied pectin solutions is extremely high, with the field of vision overrun with the ice crystals rapidly (less than in 5 seconds). For this reason, we evaluated the ice crystal melting rate in the intense melting areas for these pectin solutions.

All options for comparison of melting rates in the studied pectin solutions were subjected to analysis (Table 2). The results show that ice crystal melting rates for 0.4 wt % solutions of all studied pectins have no statistically significant difference, with the average melting rate for all solutions being 3.16 ± 0.98 μm/s. Ice crystal melting rates in the 0.2 wt % and 0.4 wt % N6HS pectin solutions and the 0.2% N7TVF and N8RS pectin solutions show no difference as well. Statistically significant differences are observed for the 0.2 wt % solutions of the following pectins: N6HS and N7TVF, N6HS and N8RS, as well as for the 0.2 wt % and 0.4 wt % N7TVF and N8RS pectin solutions.

Table 2.

The Melting Rate of Ice Crystals in Solutions of Plant Pectins (mkm/s; n = 5)

No.	Name of pectin	Melting rate
1	0.2% N6HS	2.85 ± 0.98
2	0.2% N7TVF	3.59 ± 0.88^a
3	0.2% N8RS	4.14 ± 1.13^a
4	0.4% N6HS	3.71 ± 1.38
5	0.4% N7TVF	2.88 ± 0.69^b
6	0.4% N8RS	2.82 ± 0.87^b

Significance of difference from 0.2% N6HS (p < 0.05).

Significance of difference from 0.2% (this difference when comparing different concentrations of one pectin [0.2% and 0.4%]), same pectin (p < 0.05).

Therefore, a lower ice crystal melting rate for 0.2 wt % pectin solution is identified for the N6HS pectin, that is, heracleuman. The melting rate is lower for the higher concentrations of N7TVF (tanacetan) and N8RS (rauwolfian) pectins.

The data on the integrity of cells frozen in a medium containing polysaccharides may indicate a cryoprotective effect of the N7TVF and N8RS pectins, that is, tanacetan from tansy and rauwolfian from rauwolfia (Table 3).

Table 3.

Characteristic Preservation of Leukocytes (M ± σ) Undergoing Hypothermia Effects of Temperature (−20°C) for 1 Day in Cryoprotectant Solution

Substance	n	Indicators of preservation, %
Substance	n	Amount of leukocytes	Eosin resistance	Quantity of granulocytes	Phagocytic activity of neutrophils	Content of lysosomal cationic proteins in neutrophils
Glycerol+N6HS	10	86.4 ± 13.4	75.5 ± 8.3	50.8 ± 11.9	Cells are destroyed	81.3 ± 13.2
Heracleuman+N7TVF	10	80.2 ± 7.4	77.5 ± 6.1	55.3 ± 10.2	Cells are destroyed	80.5 ± 11.9
Tanacetan+N8RS	10	85.7 ± 9.7	86.6 ± 7.2^a	65.4 ± 4.9^a	68.1 ± 7.9	94.9 ± 1.3^a
Rauwolfian	10	95.2 ± 6.8	87.0 ± 6.3^a	86.0 ± 11.0^a	78.1 ± 8.7	96.7 ± 1.8^a

Significance of difference from glycerol (p < 0.05).

Discussion

The currently accepted criterion of nucleator activity is the temperature of fluid crystallization (T_cryst), or, as it is conventionally called, the nucleation temperature. If the degree of supercooling is low, the nucleation rate will be low as well, and, vice versa, the higher the degree of supercooling, the higher the nucleation rate.

Some dissolved substances may affect the nucleation rate; moreover, the higher is the diffusion activation energy, the lower is the nucleation rate. Nucleation may never occur in highly concentrated solutions, as the components of such solution change their state completely from liquid to amorphous. Highly concentrated solutions of many cryoprotectants are capable of such amorphous solidification, including glycerol, 1.2-propanediol, ethylene glycol, saccharose, etc. The effect of these solutions is mediated through increased medium viscosity; although the crystal nuclei are intensely formed, such increased viscosity suppresses the expansion of the crystals, rendering them incapable of reaching a sufficient critical size to damage a cell.

According to our data, the highest nucleation activity was observed in heracleuman solutions (N6HS), whereas the lowest nucleation activity was registered for tanacetan solutions (N7TVF). This effect can be explained by the osmotic concentration of these solutions. Zachariassen and Kristiansen demonstrated that increased osmotic concentrations of active solutions lower the point at which biological ice nucleators cause the system to freeze.¹⁴ This correlation was illustrated by the series of tests with tanacetan solutions (N7TVF). Considering the existing data on the integrity of cells frozen in the polysaccharide-containing media, it may be concluded that polysaccharides with the lowest nucleating activity show the most promise for use as components of cryoprotectant media.

All studied polysaccharide solutions demonstrated a high linear rate of ice crystal growth. For this reason, we evaluated the ice crystal melting rate in the intense melting areas for these pectin solutions and found that there were statistically significant differences between the melting rates for the 0.2% solutions of the pectins, N6HS and N7TVF, N6HS and N8RS, as well as between concentrations for the pectin N7TVF and between concentrations for the pectin N8RS.

It was established that the macromolecule of all plant pectins comprises the main linear carbon chain of α-1.4-bound d-galacturonic acid groups with neutral monosaccharide side chains having a varying degree of branching.³⁵ It was also demonstrated that pectins extracted from various species of plants differ in the structure of the linear and branched regions, which can characterize their physical and chemical properties. The characteristic property of pectins is their ability to form viscous water solutions and gels.

As the comparative analysis of general chemical properties of the studied plant polysaccharides (Table 4) shows, the N6HS pectin has the highest content of galacturonic acid (linear region of the macromolecule) and only 7.3% of neutral monosaccharides.

Table 4.

General Chemical Characteristic of Polysaccharides Used in This Work

Name of the pectin/source isolation	GalA, %	Neutral monosaccharides, %
Name of the pectin/source isolation	GalA, %	Gal	Ara	Rha	Glc	Xyl	Man
N6HS Heracleuman/Heracleum sosnowskyi	84	2.8	2.6	1.9	—	—	—
N7TVF Tanacetan/Tanacetum vulgare	64	8.5	8.4	5.5	1.2	0.9	0.5
N8RS Rauwolfian/Rauwolfia serpentina	82	5.0	4.1	2.1	5.0	1.8	—

Ara, arabinose; Gal, galactose; GalA, galacturonic acid; Glc, glucose; Man, mannose; Rha, rhamnose; Xyl, xylose.

Source: Ovodov et al.³⁵

Galacturonic acid content in the N8RS pectin is slighly lower, but still it is 2.5 times as high as the neutral monosaccharide content. The linear region of the N7TVF pectin macromolecule contains significantly less galacturonic acid than the N6HS and N8RS pectins, whereas its neutral monosaccharide content is 3.4 times as high as that of the N6HS pectin and 1.4 times as high as that of the N8RS pectin. It can be inferred that the N6HS pectin macromolecule has the least branched side region, whereas the N6HS and N7TVF pectins have the most branched side chain. However, as the N7TVF pectin contains more neutral monosaccharides than the N8RS pectin, the N7TVF pectin is more viscous.

The osmotic and thermographic properties of the studied pectin solutions and the degree of their effects on the ice morphology formed during the phase transitions from water to ice may be attributed to structural properties of their macromolecules, namely, to the degree of side chain branching as well as quality and quantity of their neutral monosaccharides.

Therefore, plant pectin polysaccharides in the concentrations studied above have low osmotic concentration and can be expressed as the following series: 0.4 wt % N7TVF >0.4 wt % N8RS >0.4 wt % N6HS. The series according to the polysaccharide nucleation properties is as follows: N7TVF > N8RS > N6HS. Even in such low concentrations as these, pectin polysaccharides can act as modifiers of the ice morphology during the phase transitions from water to ice in cryoprotectant media. These pectins (in a concentration 0.2 wt %) were added to the formula of cryoprotective solution for evaluating their cryoprotective effect on the cells. Statistically, the best result was obtained with pectins, N7TVF and N8RS. The reason for this difference may be the ability of these pectins to modify ice, but the concentration of pectins in the cryoprotective solution is rather small and this issue requires further study.

Footnotes

Author Disclosure Statement

No competing financial interests exist.

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