DESALINATION

ELSEVIER Desalination 144 (2002) 3 13-3 18 www.elsevier.com/locate/desal

Concentration and desalination of fish gelatin by ultrafiltration and continuous diafiltration processes

Anne Simon*, Laurent Vandanjon, Guy Levesque, Patrick Bourseau Laboratoire Polymkres & Pro&d& Universite de Bretagne Sud, Rue Saint-Maudk,

BP 92116, 56321 Lorient Cedex, France Tel. +33 02.97.87.45.33; Fax +33 02.97.87.45.88; email: aelain@yahoo.com

Received 1 February 2002; accepted 15 February 2002

Abstract

Ultrafiltration (UF) of gelatin liquor from marine source (tuna skins) has been performed on a tubular mineral membrane of cut-off 10,000 Da. An attempt was made to concentrate the fish gelatin as well as to purify it by means of the diafiltration technique (DF). Primary experiments were devoted to optimise the UF performances as a function of the main adjustable parameters. The influence of pH, transmembrane pressure, recirculating velocity, and working temperature on both volumetric permeate fluxes and protein retention rates was successively examined. Under the appropriate operating conditions, it was shown that the used membrane can achieve the initial concentration step of the gelatin solutions up to 12 wt% with a protein yield as high as 0.98. In addition, continuous DF proved useful to reduce the salt content of the solutions at an average desalting speed of 185 g/h.m* with a few loss of protein in the permeate side.

Keporak: Ultrafiltration; Continuous diafiltration; Fish gelatin; Concentration; Desalting

1. Introduction gels. So far, most of the available gelatins has

Gelatin is a very important fibrous protein having numerous applications, particularly in the pharmaceutic and food industries due to its unique chemical and physical properties among which the ability to form thermally reversible

*Corresponding author.

been manufactured from mammalian resources, either pigskins or cowhides. However, since the ESB crisis, there is a growing interest for finding alternative sources of raw materials. Gelatins produced from fishery by-products are potential alternatives to mammalian gelatins [ 1,2]. Unfor- tunately, for many uses, fish gelatins obtained

Presented at the International Congress on Membranes and Membrane Processes (ICON), Toulouse, France, July 7-12, 2002.

001 l-9164/02/$- See front matter 0 2002 Elsevier Science B.V. All rights reserved PII:SOOll-9164(02)00333-S

314 A. Simon et al. /Desalination 144 (2002) 313-318

from the classical processing do not offer such good technical properties. One of the major differences for technological applications lies in the lower melting and gelling points of the gels

[2,31. In the conventional production process, after

acid or alkaline extraction from the raw mater- ials, the gelatin broths are clarified, demine- ralised by ion-exchange and then concentrated in vacuum evaporators up to 25-35 wt% gelatin.

A few works [4-S] have shown that ultra- filtration (UF) can also be successful to concen- trate mammalian gelatin solutions. Recognised advantages of this process upon the conventional evaporation method are (1) lower dewatering costs, particularly for the initial concentration stage; the energy consumption is estimated to be reduced to at least 40% [5], (2) lesser thermal degradation of the gelatin molecules since the time required ultimately in the evaporators is shorten, and (3) higher purity level for the same concentration ratio due to elimination of the impurities smaller than the nominal cut-off of the membrane.

In addition, UF can provide a further product improvement through the removal of salts from the gelatin liquors by operating in the diafil- tration mode [6,7].

Due to the above investigations, it is expected that UF may be an effective means to produce fish gelatins of upper quality.

The objective of this preliminary work was then first to assess the feasibility of using UF for the concentration and the desalting of a marine gelatin from tuna skins.

2. Materials and methods

2.1. UFpilotplant

UF tests were performed in a tubular cross- flow pilot plant of 3 L capacity (TIA, France). The UF module (50 cm’) was equipped with a commercial Al,O,-membrane MEMBRALOX@ Tl-70 (from SCT, France) having a nominal cut-

off of 10,000 Da. The same membrane was used throughout the study as its initial resistance to

flow (R,, = 3.10” m-’ at 2O’C) was entirely restored by chemical cleaning (NaOH O.lN for 45 min followed by demineralised water until neutrality) between each run.

2.2. Fish gelatin

Tuna skin gelatin was provided in a granular powder form of 87 wt% protein content. Gelatin liquors were prepared at concentration between 0.01 and 12 wt% protein by dissolving the appropriate amount of dry powder in demineral- ised water under vigourous stirring. The main characteristics of these solutions are listed in Table 1. Adjustment of pH was made by an addition of small quantities of 0.1 M NaOH or HCl. For DF experiments, sodium chloride of analytical grade (Sigma Chemical Co., St. Louis, MO, USA) was added to the ash-free gelatin to simulate the 20-30% of salts in the production liquors.

2.3. Methods

The UF unit was operated in the batch non- concentration mode keeping the initial feed volume V, at 2 L. Operating conditions were: pH 2-8.8, transmembrane pressure 60400 kPa, feed

Table 1 Principal characteristics of the fish gelatin solutions

Gel bloom’, g Mean MWb, kDa MW rangeb, kDa

PH Viscosity’, mPa.s Conductivity4, @cm-’

200 f 20 96 l-10,000 5.6-5.7 3.3 <30

“Strength of a gel formed at 6.7 wt% at 1O’C. bDetermined by gel permeation chromatography using a TSK G5000 PW column. “of a 6.7 wt% solution at 6O’C. dof a 0.1 wt’% solution before sodium chloride addition.

A. Simon et al. /Desalination 144 (2002) 313-318 315

velocity 1.4-4.7 m.s-’ and working temperature 21-55°C. Volumetric permeate fluxes were measured continuously until steady state was reached. Concentrate and permeate were regu- larly sampled for analysis of their protein content by spectrophotometric measurements at 230 nm. Retention rates were deduced from:

Retention rate = 1 - > r

where C, and C, represent the protein concen- tration (wt%) in the permeate and the retentate.

Desalting was monitored through conductivity measurements using a Crison GLP 31 con- ductivity meter.

3. Results and discussion

3. I. Ultrafiltration of gelatin liquors

Primary experiments were carried out to study the influence of the main process parameters on cross-flow ultrafiltration performances.

3.1.1. Influence of the pH of the gelatin solution

As shown in Fig. 1, steady-state flux values were found to be significantly influenced by the initial pH of the gelatin liquor. Low permeate fluxes of 20 L/h.m2 or less were achieved at the natural pH of the gelatin solution (i.e., pH 5.7) and in the entire range of pH 4 to 8.8. However, when decreasing pH below 4, fluxes changed sharply and increased by 7-8 times. Furthermore, in the latter range of pH, steady flux was attained within a few seconds while at a higher pH a sharp decrease in flux was observed during the first few minutes before a steady state was reached.

Since the isoelectric point (iep) of gelatin molecules extracted under alkaline conditions is close to pH 4-5 [9], it was suggested that the above may be explained by protein membrane fouling. This assumption was confirmed from protein adsorption experiments under static

16U 35 8 0 0

140 n n 0 30

_T‘ I20

E 25

- 100 -Gy

P 20 h

d RO m Flux z

1 60 0Rcristsnce : 15 =

k 40

20 0 OOB n m a

on..,,,.,,...,,..

195 3s 5,s 7,s 9-5

PH

Fig. 1. Effect of pH of the gelatin solution on fluxes (at C,,: 0.01%; T: 32 f 2’C; TMP: 100 kPa; U: 4.7 m.s-‘) and on the effective resistance of the membrane after fouling experiments.

solutions at pH ranging from 2 to 9. Afterwards, the hydraulic resistance of the membrane R,,, was determined from pure water flux measurements and compared to that ofthe cleaned membrane. It was observed that, below pH 4, R,,, remained almost equal to R,,,, whereas above pH 4, it became much higher, about 32.10i2 m-‘. The increase in R,,, results of gelatin adsorption on the membrane surface generating an additional resistance to flow (R,,, = R,,,, +Rads). From Fig. 1, it is obvious that the decrease in filtration rates during gelatin filtration can be ascribed to the adsorption of gelatin molecules onto the alumina membrane. Also, similar behaviour has been already quoted for mammalian gelatin [S].

Even if the accurate adsorption mechanisms have not yet been established, there is good evidence for the importance of ionic interactions with the membrane surface. Indeed, as the iep of alumina is close to pH 8-9, below pH 4 the membrane surface is positively charged as are the protein molecules. As a result, adsorption is hindered and high flux levels can be achieved. In contrast, in the pH range 4-9, both membrane and proteins are of opposite signs. In this latter case, strong electrostatic interactions can occur, thus increasing the intrinsic membrane resistance.

316 A. Simon et al. /Desalination 144 (2002) 313-318

conditions. In these experiences, the alumina membrane was soaked for 10 min into gelatin

In regard to the membrane selectivity, no effect of pH was shown. Almost perfect retention rates were found (>0.97) as expected from the membrane cut-off with respect to MW range of the fish gelatin. In the following, it was then decided to fix the pH of the feed at 3.2-3.3 to avoid membrane fouling and to maximise permeate flow rates.

It is worth noting that at this pH a slight decrease with time in the viscosity of the solution was detected which might be a sign of protein molecules denaturation. However, this variation was negligible in the duration of the cross-flow experiments.

The influence of transmembrane pressure (TMP), recirculating velocity (u) and working temperature (T) was then investigated in order to optimise the process efficiency.

3.1.2. Injluence of tangential velocity and transmembrane pressure

Examination of the effect of tangential velo- city at a constant transmembrane pressure shown that the highest permeate flux value was achieved at the highest feed velocity of 4.7 m.s-’ (Re 32,900), i.e., at the highest shear stress at the membrane wall. However, the actual flux value varied with the transmembrane pressure applied. As depicted in Fig. 2, at this optimal velocity a limiting operating pressure of 200 kPa was found. Indeed, permeate flux was shown to increase with pressure up to 200 kPa and then gradually level off, indicating that polarisation concentration became very significant beyond this TMP value. At the same time, protein retention was shown to slightly increase from 0.92 to 0.98 as a result of TMP increase from 60 kPa to 150 kPa and then to remain unaffected by further increase in pressure.

3.1.3. Injluence of operating temperature

As commonly observed in UF of proteinic

:

07 I . I . I a , ro 60 100 IS0 200 300 400

TMP (kPa)

Fig. 2. Effect of pressure on volumetric permeate flux and on membrane protein retention (C,: 0.0 1 wt%; T: 32* 2°C; pH: 3.4; u: 4.7 m.s-I).

0,Ol O,I I 10 100

Feed concentration (wt % protein)

Fig. 3. Variation of volumetric permeate flux and protein retention rate versus concentration of gelatin liquor (T: 59 2°C; pH: 3.38; PTM: 200 kPa; U: 4.7 m.s-I).

solutions, a marked benefit of higher working temperature on flux was noticed with an average rate of improvement of 2% per “C. This gain was exploited up to 55°C. However, due to the well- known heat sensitivity of gelatin proteins, it was not advisable to increase the temperature above.

3. I. 4. Permeability and selectivity of the UF membrane

Under this optimum set of working condi- tions, the process efficiency was evaluated for different protein contents of the artificial gelatin

A. Simon et al. /Desalination 144 (2002) 313-318 317

liquor. Permeate fluxes were observed to decrease from 3 10 L/h.m* to 23 L&m* as gelatin concentration increased from 0.01 w-t% to 12 wt% (Fig. 3). Such performances are as good as those reported with low gel strength mam- malian gelatin solutions in similar conditions [3,4]. Protein retention was not affected by the concentration of gelatin in the feed and was 0.98 or higher. Due to these results, it is concluded that UF cannot be envisaged for the complete dewatering of fish gelatin liquors. However, it can be an effective way to perform the initial stage of gelatin concentration up to 10-12 wt% under mild conditions in place of evaporation.

3.2. Continuous diafiltration

The efficiency of the alumina membrane in the DF mode was next evaluated for the desalting of the fish gelatin liquors. The experiences were performed on a 3.5 wt% gelatin solution with NaCl added to the salt-free fish gelatin. DF was conducted in the continuous mode, i.e., the permeate flux being compensated by an equal input of de-ionised water. In this case micro- solutes in the feed are progressively removed with the convective flux. Assuming a constant transmission of the microsolute, T,, through the membrane, the mass balance on the i-component:

- v, dCri -2 = J,,.S.C,,

dt

predicts an exponential decrease in the concentration of i with respect to p:

Cr,j = Co,i exp (-T; V *)

where Cpi, Co,,, and C,i are, respectively, the concentration of microsolutes in the retentate, in the feed and in the permeate, and V* is the diafiltration volume (ratio of volume of pure water added to initial volume of solution to be washed).

-I

n Cr,i exprimental -0.i lheoretic - 0,s

x Svlt retentioll . A Protein Mention - XI

- 0.6 8

S. s :o,4 1 Y f

95 : . 103

85 . . , . , . , .-0

0 0.2 0,4 O-6

Diivolume number V*

Fig. 4. Variation ofNaC1 concentration and of membrane retention rates for salts and proteins vs. v*. (T: 55* 2’C; pH: 3.27; PTM: 200 kPa; II: 4.7 m.s-‘, processing time: 6 h).

The recorded variation ofNaC1 concentration in the retentate and the salts and protein retention rates are reported in Fig. 4 as a function of diafiltration ratio. It can be seen that for a dia- filtration ratio of 0.6 1 (1.2 L of pure water added to the initial 2 L of gelatin liquor), the salinity of the fish gelatin was reduced by 33% (from 145 mM to 97 mM). The salt rejection was quite constant with time (about 3 5%), and thus experi- mental and theoretical variations of NaCl in the course of the DF are in good agreement. According to the model, a diafiltration ratio of 3.5 might be sufficient to remove 90% of the salts from the solution. However, with the used laboratory pilot plant such a high level of desalt- ing cannot be achieved in acceptable processing time.

It has to be pointed out that the volumetric flux remained constant throughout the run as the permeated protein loss was very low, although it was at a quite lower level than expected from UF cross-flow experiment (Fig. 3) at the same protein concentration (50 L/h.m* vs. 60 Lih.m*). Possible explanation of this lessening in flux may be the increase in the ionic strength of the solution with NaCl adjunct.

318 A. Simon et al. /Desalination 144 (2002) 3I3-318

4. Conclusions

UF membrane technology is an interesting potential adjunct to the processing of fish gelatin both to perform a first concentrating step of the gelatin liquors and to complete the desalting of these solutions. The performances, in terms of productivity, protein yield and desalting capa- bility, are similar to those reported with mammal- ian gelatin liquors.

Another potential benefit of UF process is the narrowing of the molecular mass distribution of the fish gelatin in order to improve technological and gelling properties (gel and melting temperatures, gel viscoelasticity). This aspect is now under consideration in the laboratory as well as protein chemical cross-linking.

Acknowledgements

The authors thank the Conseil Regional de Bretagne (France) for financial support and ID. Mer (Lorient, France) for providing fish gelatin.

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