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Isomerization of lactose and lactulose production: review
Trends in Food Science & Technology 18 (2007) 356e364
Review
Isomerization of lactose and lactulose production: review Mohammed Aidera,b,* and Damien de Halleuxc a
Department of Food Sciences and Technology, Laval University, Quebec G1K 7P4, Canada (Department of Food Sciences and Technology, Universite´ Laval, Pavillon Comtois, STA, Que´bec G1K 7P4, Canada; e-mail: [email protected]) b Institut National des Nutraceutiques et des Aliments Fonctionnels (INAF), Laval University, Quebec G1K 7P4, Canada; e-mail: [email protected] c Department of Food Engineering, Universite´ Laval, Pavillon Comtois, Que´bec G1K 7P4, Canada Lactulose is widely used in pharmaceutical, nutraceuticals and food industries because of its beneficial effects on human health. Technology of lactulose production is mainly based on the isomerization reaction of lactose in alkaline media. However, information available on this subject is very varied. This study is a summary of the principal techniques used for lactulose production in order to gather maximum information in one manuscript for a better comprehension of the technological characteristics and specificities of lactulose synthesis.
Introduction Significant part of the world population suffers from gastrointestinal diseases of various types. Several of these diseases are caused by pathogenic bacteria which invade the human intestine. A few days after the birth, the human intestine is colonized mainly by bifidobacteria which play a very important role in the maintenance of a good health. By changing the nutrition regime and children passage from mother’s milk nutrition to ordinary food regime, the pathogenic bacteria which infiltrated into the human * Corresponding author. 0924-2244/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.tifs.2007.03.005
intestine cause diseases of various types. In order to solve this health problem, food industry and in particular dairy technology has developed dairy bio-products enriched with probiotics like lactobacillus (Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus bulgaricus, etc.) and bifidobacteria (Bifidobacteria bifidum, Bifidobacteria longum, Bifidobacteria infantilus, Bifidobacteria adolescentis) (Clark & Martin, 1994; Donkor, Nilmini, Stolic, Vasiljevic, & Shah, in press; Katz, 2006; Ninonuevo et al., 2007; Olguin, et al., 2005; Wainwright, 2006). However, because of various reasons, this solution did not solve the problem. These reasons could be resumed by the following: a great loss of bacterial cells during the production process of different dairy products noticed by several researchers, a considerable reduction of the total of bacterial number due to storage at pH values lower than 5.5 as well as because of the strong acid medium in the stomach (pH y 1.5) and the negative effect of bile salts (Chou & Hou, 2000; Lankaputhra & Shah, 1995; Lian, Hsiao, & Chou, 2002). An alternative to the resolution of this problem consists in an internal stimulation of the bifidobacteria which are already present in the intestine (Bouhnik et al., 1990; Delzenne, 2003; Gibson, Beatty, Wang, & Cummings, 1995; Mizota, Tamura, Tomita, & Okonogi, 1987). This method consists in using bifidogenic functional food ingredients, known under general name of prebiotics (Kaplan & Hutkins, 2000; Marteau & Boutron-Ruault, 2002; Roberfroid, 2002; Saarela, Hallamaa, MattilaSandholm, & Matto, 2003; Ziemer & Gibson, 1998). These bifidogenic ingredients stimulate the growth of bifidobacteria (Tamura, Mizota, Shimamura, & Tomita, 1993). Lactulose is one of these ingredients (Alander et al., 2001; Ballongue, Schumann, & Quignon, 1997; Saarela et al., 2003). Lactulose is a synthetic disaccharide obtained by an isomerization reaction of lactose whose milk and lactoserum are very rich (Zokaee, Kaghazchi, Zare, & Soleimani, 2002). The average lactose content in milk or milk whey is approximately 4.5% (Lindmark-Mansson, Fonden, & Pettersson, 2003). Several studies showed the effectiveness of lactulose to stimulate the growth of bifidobacteria (Martin, 1996; Mizota, 1996; Sako, Matsumoto, & Tanaka, 1999; Shin, Lee, Petska, & Ustunol, 2000; Strohmaier, 1998). Moreover, lactulose is widely used in pharmaceutical industry as an effective drug against different diseases like acute and chronic constipation (Mizota, Tamura, Tomita, & Okonogi, 1987; Tamura et al., 1993).
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Taking into account all these considerations, one can deduce the great need for a large scale production of lactulose for food, nutraceuticals and pharmaceutical purposes. The raw material for this production is largely available in great quantity on the market (lactoserum as by-product of the cheese industry). Annual whey production in the world is estimated to be 72 million tons, which means that about 200,000 tons of milk proteins and 1.2 million tons of lactose are transferred into whey annually. Even though many uses of whey and some whey solids have been developed recently, only a little amount of the available whey solids are utilized as ingredients in the human nutrition and animal feed (Kosaric & Asher, 1982). Ghaly, Ramkumar, Sadaka, and Rochon (2000) estimated that in 1998 about 137.9 million tons of whey were produced in the world. As a particular case, in Canada, the annual cheese production increased by 22% between 1994 and 2004. Total cheese production in Canada in 2004 was estimated at 0.34 million tons, which implies that over 0.27 million tons of whey was produced that year (Ferchichi, Crabbe, Gil, Hintz, & Almadidy, 2005). Even though there are a multitude of technological developments in the transformation of milk whey to other useful products, utilisation or disposal of whey remains one of the most significant problem in the dairy industry (Calli & Yukselen, 2004; Mawson, 1994). Prebiotics Prebiotics are defined as non-digestible food ingredients that may beneficially affect the host by selectively stimulating the growth and/or the activity of a limited number of bacteria in the colon. Thus, to be effective, prebiotics must escape digestion in the upper gastrointestinal tract and be used by a limited number of the microorganisms comprising the colonic microflora. Prebiotics are principally oligosaccharides. They mainly stimulate the growth of bifidobacteria, for which reason they are referred to as bifidogenic factors (Durand, 1997; Berg, 1998; Gibson & Roberfroid, 1995; Macfarlane & Cummings, 1999; Roberfroid, 2000). So that a food ingredient can be regarded as prebiotic, it must meet certain characteristics which were defined gradually after the initial work of Gibson and Roberfroid (1995). Food ingredient can be regarded as prebiotic if it satisfies some criteria (Gibson & Roberfroid, 1995): not digestible nor absorbed before reaching the colon; to be a selective substrate of one or several (preferably a low number) bacteria having a probable or definitively established beneficial role; to be able to modify the composition of the colic flora for better health by supporting the growth and/or the metabolic activity of Lactobacillus sp. or Bifidobacteria sp. (Gibson & Roberfroid, 1995); more rarely by attenuating the virulence of pathogenic bacteria like Listeria monocytogenes (Park & Kroll, 1993). Some researchers reported some information, where the role of the prebiotics is not totally clear. In was postulated
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that prebiotic ingestion may contribute to normalize the gastrointestinal barrier function in burn patients (Olguin et al., 2005). This hypothesis was based on observations that burn injury is associated with dramatic alterations of the intestinal microbiota and gastrointestinal permeability, and that increasing luminal lactobacilli and bifidobacteria through the ingestion of prebiotics or probiotics is associated with recovery of the gastrointestinal barrier function. This postulate was based on the observation that regular intake of Lactobacillus spp. decreased the gastric permeability alterations. In relation to burn injury, a decrease of the intestinal anaerobic microbiota, including bifidobacteria, has been observed in rats, while at the same time aerobic bacteria and fungi increase. This resulted in an imbalance of the aerobic/anaerobic ratio and in a decrease of colonization resistance in these animals. These changes were associated with increased bacterial translocation and endotoxinemia, histological lesions of the mucosa. Similar alterations have been observed in burn patients. Supplementation of burn rats with a bifidobacteria preparation reduced the imbalance of the aerobic/aerobic ratio, the endotoxinemia and the mucosal lesions; the same preparation with bifidobacteria decreased gastrointestinal symptomatology and diarrhea in humans who suffered burns (Chen, Zhang, & Xiao, 1998; Gotteland, Cruchet, & Verbeke, 2001; Olguin et al., 2005). Stimulation of endogenous lactobacilli or bifidobacteria by prebiotics may also exert a protective effect against gastrointestinal mucosa alterations. Lactosucrose, for example, has been shown to protect against indomethacin-induced gastric ulcerations in rats (Honda et al., 1999). Although a number of studies have been carried out in animal models, data are scarce in humans. In the study reported by Olguin et al. (2005), oligofructose, whose administration is known to dosedependently increase fecal bifidobacteria in humans, was used. This prebiotic did not improve the gastrointestinal barrier function alterations. A possible explanation for this lack of effect is the use of high doses of antibiotics in all these patients, which may interfere with lactobacilli and bifidobacteria growth even after stimulation by the administrated prebiotic (oligofructose). In the case of probiotics, this may be overcome by the continuous administration of these exogenous, live bacteria which may compensate for the mortality induced by antibiotics; prebiotics, however, act by stimulating the growth of endogenous bacteria, and this is probably decreased when these microorganisms are affected by antibiotics. The results obtained in the above mentioned studies may be interpreted as suggesting that prebiotics probably are not the best option for subjects on high doses of antibiotics, and that administration of probiotics or symbiotic, a mixture of pre and probiotics may be a better choice for these patients (Olguin et al., 2005). However, even if the used prebiotic showed some negative aspects, we can not generalise this conclusion to all the prebiotics, including lactulose. The use of antibiotics would be the
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cause of the negative effect reported in the study of Olguin et al. (2005). Lactulose production Theoretical aspect of lactulose production Theoretically, lactulose (Fig. 1) can be obtained starting from lactose (Kochetkov & Bochkov, 1967) by regrouping the glucose residue to the fructose molecule with a passage form an aldose form to ketosis one. The mechanism of this transformation can be achieved by various manners. The first consists of the reaction of Lobry de Bruynevan Ekenstein which is summarized by the formation of the enolic intermediate shape of lactose and epilactose in alkaline media with the transformation of the glucose of the lactose molecule into fructose and gives as result molecule of lactulose (Fig. 2). The second way consists of a reaction of lactose with ammonia or amines. In this case, the formed lactosylamine undergoes a regrouping (rearrangement) of Amadori (Fig. 3) towards the lactulosylamine (Hodge, 1955) and after hydrolysis of the complex lactulose could be obtained. Currently, in practice, the first way of synthesis is the most used with various catalysts. The energy of activation for synthesis of lactulose by using lactose as raw material differs according to the type of the catalyst (European Patent No 0320670, 1990; European Patent No 0339749, 1991; U.S. Patent No 5034064, 1991). To transform lactose into lactulose, acceptors of protons are essential. This could be carried out by using various reagents which give an alkaline medium after dissolution (European Patent No 0339749, 1991; U.S. Patent No 3814174, 1971; U.S. Patent No 5034064, 1991; U.S. Patent No 4536221, 1985; U.S. Patent No 3514327, 1970; U.S. Patent No 3546206, 1970). The great number of reagents used shows well that the ideal catalyst was not found yet for the isomerization of lactose to lactulose. This catalyst must answer some important criteria, among which are enumerated by the following: It must guarantee a maximum level of isomerization with a minimum of reaction by-products;
To be environmentally safe and not toxic; The cost of the catalyst must be as possible low and to be available in great quantity; It must be easy to remove from the medium by traditional demineralization tools; To give repetitive results of isomerization. However, in practice, the catalysts used for the isomerization of lactose to lactulose present positive and negative aspects. Systematic analysis of the most used catalysts for lactulose production could be divided into three principal groups. They are strong acids, strong bases and amphoteric catalysts represented mainly by hydroxyls, sulphites and borates. Principal methods as well as the reactions which control the process of isomerization of lactose into lactulose will be treated in what follows. Lactose isomerization by hydroxyls Lactulose was obtained for the first time by Montgomery and Hudson (1930) following the heating of a solution made up of a mixture of lactose and lime at a temperature of 35 S during several days. In order to obtain crystalline lactulose, several stages of purification were used. Reagents such as sulphuric acid, calcium carbonate, ethanol, methanol, ether, activated carbon and bromine were used. Thereafter, a new method was developed (Matvievsky, 1979; Russian Patent No 1392104, 1988; U.S. Patent No . 3272705, 1966; Yakovleva, 1963) in which calcium hydroxide was used as catalyst of the isomerization reaction of lactose to lactulose. In this method, 60% lactose solution was combined with 0.1% of calcium hydroxide under a temperature of 100e102 C and reaction time of 15 min. Thereafter, final solution was demineralised by combination of electrolysis and ion exchange resins. However, this method was rather tiresome. In the work reported in Patent of Germany No 222468033 (1976), the use of various catalysts such as (Ca(OH)2, NaOH, CaO, Na2CO3, KOH, K2HPO4, Ba (OH)2) was reported (Dalev & Tsoneva, 1982; Lodigin, 1999; Patent of Germany No 297999, 1992; Pereligin, Podgornov, & Sitnikov, 1999; Russian Patent No
Fig. 1. Schematic representation of lactulose molecule.
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Fig. 2. Representation of the Lobry de Bruynevan Ekenstein transformation.
2101358, 1998; Russian Patent No 97120603/13, 1999; Stomberg & Semchenko, 1999; U.S. Patent No 4264763, 1978). In this study, effect of these catalysts on galactose rate as by-product of disaccharides (lactose and/or lactulose) decomposition (degradation) was also reported. While being based on results reported in Patent of Germany No 222468033 (1976), the following conclusions could be mentioned: Independently on the quantity of NaOH used, there exists a linear dependence between galactose rate produced as reaction by-product and lactulose rate which represents rate the reaction isomerization rate. This relation seems to increase considerable at isomerization rates higher than 21.4%; Galactose/lactulose ratio remains constant for a constant ratio NaOH/lactose independently on isomerization time and temperature. Basing on results reported in Patent of Germany No 222468033 (1976), an industrial application was carried out (France Patent No 2147925, 1973; Patent of Germany No 222468033, 1976). In the process used for the industrial production, lactulose was purified and demineralised by anion and cation exchange resins. The end product had a concentration of the lactulose of about 42e48% of total dry matter. However, even if this method was effective, it remains that it needed rather important material and energy expenditure. Another method was developed in Russian patent No 7374626 (1980). In this method, 15e20% of lactose solution was mixed with 0.35e0.45% of the calcium hydroxide to reach pH 11. The mixture was thermostated
Fig. 3. Schematization of the Amadori rearrangement.
at 68e72 S during 15e20 min. The final solution pH was 8.8e9.0. Thereafter, solution was neutralized with citric acid up to pH 5.5e6.5. Citric acid quantity added in a form of saturated solution was 0.115e0.125%. The pH decrease was carried out to avoid an autocatalytic degradation of lactulose and to facilitate partial demineralization of final solution by means of calcium citrate formation (calcium complexation) removed by centrifugation. An additional operation of demineralization with of ions exchange resins was necessary. Effect of lactose concentration. According to fundamental laws of chemical kinetics, the lactose isomerization process into lactulose depends on lactose concentration in the feed solution. Systematic analysis of data on lactulose production showed significant variability of this parameter and it varies in the range of 5e60%. Moreover, the choice of lactose optimal concentration in the feed solution depends mainly on type of the catalyst. Using hydroxides (sodium, potassium or calcium) as catalysts and in order to determine the optimal lactose concentration giving maximum lactulose reaction rate with minimum coloration of the final solution, experiments with lactose concentrations varied between 5% and 30% were carried out (Ryabtsova, 1992). Lactose used in this work was of food grade but not refined. It was purified from proteins to avoid reaction between lactose and protein amine groups. Isomerization reaction was carried out by using sodium hydroxide as catalyst with an initial solution pH of 11.0 0.2 under 70 2 S and reaction time of 20 2 min. Data reported by Ryabtsova (1992) showed that lactose concentration in the range of 5e30% did not have any significant effect on the reaction isomerization rate. However, these same data showed that at lactose initial concentration higher than 20%, reaction by-products rate was higher then when lower lactose concentration were used. Same results were reported with calcium hydroxide as catalyst. Moreover, to avoid lactose crystallization after cooling the solution, it is important to use concentration in the range of 20e25%. In one other study (Montilla, del Castillo, Sanz, & Olano, 2005); concentrates of whole whey and ultrafiltrates were used to produce lactulose. Whole milk whey and milk
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whey ultrafiltrate concentrated up to 5.2 and 7.6 fold were used. Results indicated that the amount of produced lactulose increased by increasing the concentration factor of the raw material. Indeed, using simple milk, 0.388 and 1.17 g/100 ml were obtained after 30 and 120 min, respectively, of treatment. By concentrating the whey with a concentration factor up to 5.2 fold, the amounts of produced lactulose were 3.41 and 4.80 g/100 ml after 30 and 120 min, respectively. Further concentration of the initial milk whey up to a concentration factor of 7.6 fold permitted to obtain lactulose concentrations of 5.33 and 7.15 g/100 ml after 30 and 120 min of treatment (Montilla et al., 2005).
Effect of lactose and catalyst concentrations on solution pH. In the case of lactose isomerization into lactulose, solution pH characterizes concentration of proton acceptors and it plays a very important role and could be regarded as one of the more influencing factors on the isomerization reaction of lactose into lactulose. For this reason, combined effect of lactose concentration and alkaline catalyst on pH of the medium was studied by several authors. Also, this aimed to determine optimal amount of the catalyst to maintain solution pH in favourable interval for a maximum isomerization with a minimal rate of the reaction byproducts. Experimental results showed that there is no significant difference between pH values of model solutions composed by lactose of high purity compared with refined lactose solutions. Moreover, analysis of experimental data showed that independently on lactose concentration, curve of solution pH evolution shows logarithmic behavior. During first reaction stage, solution pH abruptly increases and it was reported that to increase pH of lactose solution from 5.5 0.5 up to 9.0 0.2, NaOH concentration needed is the same one independently on lactose concentration. In [100], NaOH concentration needed to increase pH from 5.5 0.5 up to 9.0 0.2 is 0.004 0.001 M. At the second stage of pH curve evolution, increase of pH was very low. During period, lactose concentration had significant effect on pH increase. It is more difficult to increase the pH of the concentrated lactose solutions. For example, to increase pH up to 11.0e11.5 of lactose solution of concentration of 0.015e0.03 M it was necessary to add 0.03e0.05 M of NaOH and for lactose solution of a concentration of 0.06 M, NaOH concentration of 0.06e0.07 M was needed. In dilute media, no significant difference was reported on NaOH concentration needed to increase pH from 5.5 0.5 up to 11.5 0.1 between raw and refined lactose. But, for lactose concentration above 0.06 M, to reach the same pH (11.5 0.1), it is necessary to use more NaOH in the case of raw lactose then with refined lactose. This difference is caused by the presence of minerals and nitrogenized compounds with high buffer capacity. Similar results as those obtained with NaOH were reported when Ca(OH)2 was used as catalyst of the isomerization reaction
of lactose into lactulose. To reach pH value of 11.0 0.2, concentrations of 0.02e0.04 and 0.05.0.06 M of Ca(OH)2 were added to 0.015e0.03 and 0.06 N lactose solutions, respectively (Ryabtsova, 1992). As general conclusion on relationship between lactose solution concentration and catalyst concentration, it could be resumed as follows: at pH 11.0 for each 1 M NaOH it needs 10 M of model solution lactose independently on concentration; 3e7 M NaOH in the case of concentrated lactose solution from milk whey dependently on the cheese process and finally 1e2 M NaOH for lactose solution made from raw lactose of high quality. So, we can see that by increasing lactose solution buffer capacity, lactose/catalyst ratio decreases. In general, it is important to know purity of lactose for a good choice of an optimal catalyst concentration because differences between results obtained with model lactose solutions and real solutions are significant (Ryabtsova, 1992). It was also reported that during the isomerization of lactose to lactulose with sodium hydroxide, a high level of degradation occurred and to decrease the amount of the formed reaction by-products, it is better to use the lower ratios of sodium hydroxide to lactose (about 0.5% w/w). In this case the maximum conversion of lactose to lactulose was about 20% and total by-product is about 5e7% (Zokaee et al., 2002).
Particularities of lactose isomerization by sodium and calcium hydroxide. Isomerization of lactose into lactulose with NaOH as catalyst of the reaction was carried out by using raw lactose solution of high quality at a concentration of 20%. Initial solution pH was fixed at 11.0 0.2. Lactose solution was thermostated in batch mode at 70 C. Samples were analyzed for optical density, pH evolution and isomerization rate which was represented by lactose/lactulose ratio. Experimental data reported by Ryabtsova (1992) showed that at the first stage of the isomerization reaction, there was a considerable growth of lactulose rate with a light change of solution color which characterizes formation of reaction by-products. The second reaction stage was characterized by a stability followed by a decrease of the isomerization rate. This was more intense when pure lactose solutions were used. During this stage, the decrease pH was significant and followed by an intensification of the solution color. The pH decrease was also intensified by the formation of reaction by-products with an acid character. Decrease of solution pH is an indication of the decease of proton acceptors concentration which in its turn diminishes the probability of intramolecular regrouping of lactose and its isomerization into lactulose. Moreover, because of pH decrease, lactulose degradation resulting in galactose and fructose formation could be accelerated. Another phenomenon is also possible following pH decrease which is the reversible isomerization of the lactose into lactulose through an enolic form. Same results were reported with calcium hydroxide as catalyst
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of lactose isomerization reaction. However, calcium hydroxide intensification of solution mixing is important to avoid precipitation of the catalyst. In general, it appears from systematic data analysis that maintenance of high alkalinity of the medium is an essential condition for an optimal isomerization rate of lactose into lactulose.
Formation of isomerization reaction by-products. Analysis of data on the variation of optical density and pH during degradation of sugars in alkaline media showed existence of three stages, which are closely related to the stages of lactulose formation during lactose isomerization. The first stage corresponds to a minimal degradation and this period corresponds to the highest level of lactulose formation. The second stage corresponds to the growth of sugars degradation by-products. This stage is followed by pH decrease and lactulose formation. Finally, a third stage which is characterized by a stabilization of solution color growth even at relatively high reaction temperature (72 2 C). During this third stage, pH decrease could reach neutrality. However, even if measurement of solution optical density could not give sufficient information about rate and nature of the formed dyes following lactose and/or lactulose degradation, it remains that this information could be used for better planning of lactulose production process, especially with regard to stages of residual lactose crystallization and lactulose refining which is the end product of the process (Rudenko, 1999; Rudenko & Bobrovnik, 1999). In order to better understand the process of color growth (dye formation) during isomerization of lactose into lactulose, 20% lactose solution was studied with sodium and calcium hydroxide as catalyst under temperature of 72 2 C. Results reported by Ryabtsova (1992) on optical density measurements showed that in the region of visible spectrum, growth of the absorbance was linear and that the maximum was reported to a wavelength of 360 nm. By increasing the reaction time, highly significant increase of the mixture optical density was recorded. This could be explained by the fact that in the zone of visible spectrum, different functional groups of dyes products have identical absorbance spectra which differ only by intensity as reported in Sapronov (1975). By increasing reaction time with sodium hydroxide as catalyst, a spectrum of absorbance to 490 nm was shifted and this could correspond to a change of the ratio between reaction by-products. The same data were reported using calcium hydroxide as catalyst but with a longer reaction time compared to NaOH. Optical density measurements of 0.5% lactose mixed with lactulose solution in UV zone showed maximal absorbance at 270e280 nm, which is a zone of the absorbance of lactose. Once the mixed solution of lactose/lactulose was thermostated under temperature of 72 2 C, the shape of the initial spectrum had changed significantly and other spectra of absorbance appeared. By increasing reaction
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time, maximum of absorbance was moved towards 260e 270 nm, which corresponds to the zone of absorbance of lactose/lactulose degradation by-products in alkaline media. Study of UV spectrum during lactose isomerization into lactulose could be used as base to confirm that the growth color in solution under reaction could be caused by degradation of reducing sugars when NaOH was used as catalysts (Parrish, Hicks, & Doner, 1980).
Isomerization of lactose by sulphites and phosphates Sulphites and phosphates have the characteristic to prevent oxidation of disaccharides and for this reason their use as catalyst of lactose isomerization reaction into lactulose allows the use of high temperatures and high lactose concentrations. According to data reported in Patent of Austria No 288595 (1971), for lactulose production, lactose solutions of 60e65% were used and temperature of 80e100 S. Under these conditions, sulphites were added at a rate of 0.05e0.05 M per kg of lactose monohydrate. Thereafter, the mixture (lactose solution with catalyst) was thermostated at this temperature until obtaining a constant value of the optical rotation of the solution. Then, the solution was cooled followed by crystallization of part of residual lactose. After crystallization, the solution of lactose/lactulose was treated by ion exchange resins for purification from sulphites (catalyst) and organic acids. Following this operation, another part of lactose was crystallized by cooling. To accelerate the crystallization process of lactose, it was recommended to add a sowing in the form of fine lactose crystals for a maximum crystallization yield (Polyansky & Shestov, 1995). The end product was syrup with 54.5% of dry matter, 38.7% of the lactulose. Galactose and lactose contents in the final product were about 8.2% and 3.8%, respectively. Another method for lactulose production was reported in Patent of Great Brittan No 2031430 (1980). According to the method described by the authors, 2.1e8.6% of phosphates was added to a saturated lactose solution. The temperature of the reaction was 104 S during 20e240 min, dependently of the ratio lactose/catalyst. In this case, maximum isomerization rate reported was 20%. At the end of reaction time, part of lactose was crystallized by cooling and was removed from the medium by filtration. The remainder solution was diluted up to 15% and treated by anion and cation exchange resins for purification from organic acids formed as reaction byproducts and phosphate (catalyst). After this operation, the solution was concentrated another time and part of lactose was removed by crystallization. Other methods were also reported (U.S. Patent No 4536221, 1985). The catalyst used in these cases was a mixture of sodium hydroxide and sodium sulphite at a concentration of 0.3e1% with 60% lactose solution under a temperature of 75e80 S during 15 min. The isomerization rate reported reached 30%. At the other hand, using 0.7% of sodium sulphite as catalyst, isomerization rate reached 40%. In the patent described
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in U.S. Patent No 4536221 (1985), the use of magnesium hydroxide mixed with sodium hydrosulphide in lactose solution of 60e70% concentration was reported. The catalyst concentration in this case was 0.05e0.2% and the temperature used was 90e100 S.
Isomerization of lactose by aluminates and borates Using amphoteric electrolytes such aluminium hydroxide or boric acid, total isomerization yield of lactose into lactulose could reach 70e80% (European Patent 0320670, 1990; Mendicino, 1960; U.S. Patent 4273922, 1981). In these cases, the catalyst must be added at a rate of 0.5e4 M per mole of lactose as reported in the US patent No 4957564. In U.S. Patent No 3546206 (1970), it was reported that catalyst (aluminate) was added to lactose solution. Thereafter, the mixture was heated and thermostated a certain time and then the catalyst was removed from the medium using crystallization by cooling. The pH was readjusted with HCl or aluminium hydroxide, dependently on the case. At the end of the process, it was recommended to isolate lactulose using methanol. Carrobi and Innocenti (1990) proposed using of membrane to remove catalyst after isomerization. Following this work, patent was deposited (U.S. Patent No 4957564, 1990). In these patents, it was reported that 25e50% lactose solution was used. The catalyst (sodium aluminate) was added in the form of 35e45% concentration solution. The ratio aluminate/lactose was 0.3/1 up to 1/1, dependently on lactose concentration. Isomerization was carried out under a temperature of 50e70 C during 30e60 min. At the end of the reaction time, solution was neutralized with 3e4 N sulphuric acid to keep pH in the range of 4.5e8.0. Aluminium hydroxide suspension was formed and then removed from the medium by centrifugation followed by membrane treatment. Other authors reported the use of sodium tetraborate, sodium hydroxide or triethylamine mixed with boric acid as catalyst (Hicks, Raupp, & Smith, 1984; Mizota et al., 1987). Crystallization, pasteurization and purification operations by ion exchange resins were necessary.
Isomerization of lactose by alkaline-substituted sepiolites De la Fuente, Jua´rez, de Rafael, Villamiel, and Olano (1999) reported that strong base catalysts, prepared by substituting a part of the Mg2þ located at the borders of the channels of sepiolite with alkaline ions (Liþ, Naþ, Kþ and Csþ), were investigated as catalysts for the isomerization of lactose to lactulose and epilactose. The activities exhibited by alkaline-exchanged sepiolites were significantly higher than that of natural sepiolite. The influence of temperature, time of the reaction and catalyst loading were also evaluated. A 20% conversion was obtained at 90 C at a catalyst loading of 15 g/l. At the other hand, Villamiel, Corzo, Foda, Montes, and Olano (2002) reported
that alkaline-substituted (Naþ, Kþ) sepiolites were used as catalysts for the formation of lactulose in milk permeate. Besides lactose and lactulose, other carbohydrates, such as galactose and epilactose, produced in side reactions, were determined. The effect of different washing cycles of sepiolite on the isomerization of lactose and the exchange of cations with the permeate was also investigated. In general, the activity of the sodium sepiolite was higher than that of potassium form. Twenty per cent of lactulose formation (1000 mg/100 ml), with respect to the initial lactose, was obtained after 150 min of reaction, using sodium sepiolite washed during 10 cycles. Under these conditions, 25% of lactose degradation was detected, whereas small amounts of epilactose and galactose were formed. The exchange of Naþ between sepiolite and permeate decreased considerably with the number of washing cycles. The present work shows an appropriate method for obtaining lactulose in milk permeate with acceptable yields and without complicated purification steps. Lactulose production by ion exchange resins Production of lactulose in a form of concentrated solutions or powder for use as additive in functional food is more and more of topicality. This type of product must be at the same time safe and easy to produce, whether for environment or human consumption. Based on this concept, several works were carried out during the 10 last years in order to find effective technologies for lactose isomerization into lactulose without use of reactive agents. As it was mentioned above, lactulose production requires use of chemicals (hydroxide, borates, aluminates, etc.) and purification procedures by ion exchange resins. In the Russian Patent No 2101358 (1998), anion exchange resins were used to intensify process isomerization of lactose into lactulose by exploiting OH ions exchange between solution in reaction and resins and to simplify lactulose production process by using the same resins for demineralization of the end product. The advantages of this process are There is no need to add catalyst for isomerization process; Demineralization stage is not used; No additional operation to purify the end product from dyes; He process is more profitable in comparison with traditional methods; Lactulose produces by this technology could be used in functional food for children, and in specialized food into which bifidobacteria are introduced (Hramtsov et al., 2004). Concluding remarks Lactulose production is a complex process, which is affected by several operation and technological conditions.
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Most of the changes incurred to the lactulose by heating are disadvantageous to quality of the final product. However, degradations can be minimized by appropriate design of the isomerization process, type of catalyst used and lactose quality. Designing process of lactose isomerization into lactulose must be done in a comprehensive way considering pre-isomerization, isomerization and postisomerization processes. Isomerization of lactose into lactulose must be preceded by adequately chosen raw material yields with expected quality and optimal lactose/ catalyst ratio. That quality can be maintained during lactulose isolation by application of appropriate post-processing parameters. Because of complex influence on the product and many technological variables, which can be controlled during processing, isomerization is a versatile way to treatlactose, which is an abundant product of cheese processing. A thorough knowledge of pros and cons of the isomerization process is needed in order to design optimal technological process for lactose isomerization into lactulose with a minimum of by-products and to obtain final product of desired quality for a variety of use.
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Review
Isomerization of lactose and lactulose production: review Mohammed Aidera,b,* and Damien de Halleuxc a
Department of Food Sciences and Technology, Laval University, Quebec G1K 7P4, Canada (Department of Food Sciences and Technology, Universite´ Laval, Pavillon Comtois, STA, Que´bec G1K 7P4, Canada; e-mail: [email protected]) b Institut National des Nutraceutiques et des Aliments Fonctionnels (INAF), Laval University, Quebec G1K 7P4, Canada; e-mail: [email protected] c Department of Food Engineering, Universite´ Laval, Pavillon Comtois, Que´bec G1K 7P4, Canada Lactulose is widely used in pharmaceutical, nutraceuticals and food industries because of its beneficial effects on human health. Technology of lactulose production is mainly based on the isomerization reaction of lactose in alkaline media. However, information available on this subject is very varied. This study is a summary of the principal techniques used for lactulose production in order to gather maximum information in one manuscript for a better comprehension of the technological characteristics and specificities of lactulose synthesis.
Introduction Significant part of the world population suffers from gastrointestinal diseases of various types. Several of these diseases are caused by pathogenic bacteria which invade the human intestine. A few days after the birth, the human intestine is colonized mainly by bifidobacteria which play a very important role in the maintenance of a good health. By changing the nutrition regime and children passage from mother’s milk nutrition to ordinary food regime, the pathogenic bacteria which infiltrated into the human * Corresponding author. 0924-2244/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.tifs.2007.03.005
intestine cause diseases of various types. In order to solve this health problem, food industry and in particular dairy technology has developed dairy bio-products enriched with probiotics like lactobacillus (Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus bulgaricus, etc.) and bifidobacteria (Bifidobacteria bifidum, Bifidobacteria longum, Bifidobacteria infantilus, Bifidobacteria adolescentis) (Clark & Martin, 1994; Donkor, Nilmini, Stolic, Vasiljevic, & Shah, in press; Katz, 2006; Ninonuevo et al., 2007; Olguin, et al., 2005; Wainwright, 2006). However, because of various reasons, this solution did not solve the problem. These reasons could be resumed by the following: a great loss of bacterial cells during the production process of different dairy products noticed by several researchers, a considerable reduction of the total of bacterial number due to storage at pH values lower than 5.5 as well as because of the strong acid medium in the stomach (pH y 1.5) and the negative effect of bile salts (Chou & Hou, 2000; Lankaputhra & Shah, 1995; Lian, Hsiao, & Chou, 2002). An alternative to the resolution of this problem consists in an internal stimulation of the bifidobacteria which are already present in the intestine (Bouhnik et al., 1990; Delzenne, 2003; Gibson, Beatty, Wang, & Cummings, 1995; Mizota, Tamura, Tomita, & Okonogi, 1987). This method consists in using bifidogenic functional food ingredients, known under general name of prebiotics (Kaplan & Hutkins, 2000; Marteau & Boutron-Ruault, 2002; Roberfroid, 2002; Saarela, Hallamaa, MattilaSandholm, & Matto, 2003; Ziemer & Gibson, 1998). These bifidogenic ingredients stimulate the growth of bifidobacteria (Tamura, Mizota, Shimamura, & Tomita, 1993). Lactulose is one of these ingredients (Alander et al., 2001; Ballongue, Schumann, & Quignon, 1997; Saarela et al., 2003). Lactulose is a synthetic disaccharide obtained by an isomerization reaction of lactose whose milk and lactoserum are very rich (Zokaee, Kaghazchi, Zare, & Soleimani, 2002). The average lactose content in milk or milk whey is approximately 4.5% (Lindmark-Mansson, Fonden, & Pettersson, 2003). Several studies showed the effectiveness of lactulose to stimulate the growth of bifidobacteria (Martin, 1996; Mizota, 1996; Sako, Matsumoto, & Tanaka, 1999; Shin, Lee, Petska, & Ustunol, 2000; Strohmaier, 1998). Moreover, lactulose is widely used in pharmaceutical industry as an effective drug against different diseases like acute and chronic constipation (Mizota, Tamura, Tomita, & Okonogi, 1987; Tamura et al., 1993).
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Taking into account all these considerations, one can deduce the great need for a large scale production of lactulose for food, nutraceuticals and pharmaceutical purposes. The raw material for this production is largely available in great quantity on the market (lactoserum as by-product of the cheese industry). Annual whey production in the world is estimated to be 72 million tons, which means that about 200,000 tons of milk proteins and 1.2 million tons of lactose are transferred into whey annually. Even though many uses of whey and some whey solids have been developed recently, only a little amount of the available whey solids are utilized as ingredients in the human nutrition and animal feed (Kosaric & Asher, 1982). Ghaly, Ramkumar, Sadaka, and Rochon (2000) estimated that in 1998 about 137.9 million tons of whey were produced in the world. As a particular case, in Canada, the annual cheese production increased by 22% between 1994 and 2004. Total cheese production in Canada in 2004 was estimated at 0.34 million tons, which implies that over 0.27 million tons of whey was produced that year (Ferchichi, Crabbe, Gil, Hintz, & Almadidy, 2005). Even though there are a multitude of technological developments in the transformation of milk whey to other useful products, utilisation or disposal of whey remains one of the most significant problem in the dairy industry (Calli & Yukselen, 2004; Mawson, 1994). Prebiotics Prebiotics are defined as non-digestible food ingredients that may beneficially affect the host by selectively stimulating the growth and/or the activity of a limited number of bacteria in the colon. Thus, to be effective, prebiotics must escape digestion in the upper gastrointestinal tract and be used by a limited number of the microorganisms comprising the colonic microflora. Prebiotics are principally oligosaccharides. They mainly stimulate the growth of bifidobacteria, for which reason they are referred to as bifidogenic factors (Durand, 1997; Berg, 1998; Gibson & Roberfroid, 1995; Macfarlane & Cummings, 1999; Roberfroid, 2000). So that a food ingredient can be regarded as prebiotic, it must meet certain characteristics which were defined gradually after the initial work of Gibson and Roberfroid (1995). Food ingredient can be regarded as prebiotic if it satisfies some criteria (Gibson & Roberfroid, 1995): not digestible nor absorbed before reaching the colon; to be a selective substrate of one or several (preferably a low number) bacteria having a probable or definitively established beneficial role; to be able to modify the composition of the colic flora for better health by supporting the growth and/or the metabolic activity of Lactobacillus sp. or Bifidobacteria sp. (Gibson & Roberfroid, 1995); more rarely by attenuating the virulence of pathogenic bacteria like Listeria monocytogenes (Park & Kroll, 1993). Some researchers reported some information, where the role of the prebiotics is not totally clear. In was postulated
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that prebiotic ingestion may contribute to normalize the gastrointestinal barrier function in burn patients (Olguin et al., 2005). This hypothesis was based on observations that burn injury is associated with dramatic alterations of the intestinal microbiota and gastrointestinal permeability, and that increasing luminal lactobacilli and bifidobacteria through the ingestion of prebiotics or probiotics is associated with recovery of the gastrointestinal barrier function. This postulate was based on the observation that regular intake of Lactobacillus spp. decreased the gastric permeability alterations. In relation to burn injury, a decrease of the intestinal anaerobic microbiota, including bifidobacteria, has been observed in rats, while at the same time aerobic bacteria and fungi increase. This resulted in an imbalance of the aerobic/anaerobic ratio and in a decrease of colonization resistance in these animals. These changes were associated with increased bacterial translocation and endotoxinemia, histological lesions of the mucosa. Similar alterations have been observed in burn patients. Supplementation of burn rats with a bifidobacteria preparation reduced the imbalance of the aerobic/aerobic ratio, the endotoxinemia and the mucosal lesions; the same preparation with bifidobacteria decreased gastrointestinal symptomatology and diarrhea in humans who suffered burns (Chen, Zhang, & Xiao, 1998; Gotteland, Cruchet, & Verbeke, 2001; Olguin et al., 2005). Stimulation of endogenous lactobacilli or bifidobacteria by prebiotics may also exert a protective effect against gastrointestinal mucosa alterations. Lactosucrose, for example, has been shown to protect against indomethacin-induced gastric ulcerations in rats (Honda et al., 1999). Although a number of studies have been carried out in animal models, data are scarce in humans. In the study reported by Olguin et al. (2005), oligofructose, whose administration is known to dosedependently increase fecal bifidobacteria in humans, was used. This prebiotic did not improve the gastrointestinal barrier function alterations. A possible explanation for this lack of effect is the use of high doses of antibiotics in all these patients, which may interfere with lactobacilli and bifidobacteria growth even after stimulation by the administrated prebiotic (oligofructose). In the case of probiotics, this may be overcome by the continuous administration of these exogenous, live bacteria which may compensate for the mortality induced by antibiotics; prebiotics, however, act by stimulating the growth of endogenous bacteria, and this is probably decreased when these microorganisms are affected by antibiotics. The results obtained in the above mentioned studies may be interpreted as suggesting that prebiotics probably are not the best option for subjects on high doses of antibiotics, and that administration of probiotics or symbiotic, a mixture of pre and probiotics may be a better choice for these patients (Olguin et al., 2005). However, even if the used prebiotic showed some negative aspects, we can not generalise this conclusion to all the prebiotics, including lactulose. The use of antibiotics would be the
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cause of the negative effect reported in the study of Olguin et al. (2005). Lactulose production Theoretical aspect of lactulose production Theoretically, lactulose (Fig. 1) can be obtained starting from lactose (Kochetkov & Bochkov, 1967) by regrouping the glucose residue to the fructose molecule with a passage form an aldose form to ketosis one. The mechanism of this transformation can be achieved by various manners. The first consists of the reaction of Lobry de Bruynevan Ekenstein which is summarized by the formation of the enolic intermediate shape of lactose and epilactose in alkaline media with the transformation of the glucose of the lactose molecule into fructose and gives as result molecule of lactulose (Fig. 2). The second way consists of a reaction of lactose with ammonia or amines. In this case, the formed lactosylamine undergoes a regrouping (rearrangement) of Amadori (Fig. 3) towards the lactulosylamine (Hodge, 1955) and after hydrolysis of the complex lactulose could be obtained. Currently, in practice, the first way of synthesis is the most used with various catalysts. The energy of activation for synthesis of lactulose by using lactose as raw material differs according to the type of the catalyst (European Patent No 0320670, 1990; European Patent No 0339749, 1991; U.S. Patent No 5034064, 1991). To transform lactose into lactulose, acceptors of protons are essential. This could be carried out by using various reagents which give an alkaline medium after dissolution (European Patent No 0339749, 1991; U.S. Patent No 3814174, 1971; U.S. Patent No 5034064, 1991; U.S. Patent No 4536221, 1985; U.S. Patent No 3514327, 1970; U.S. Patent No 3546206, 1970). The great number of reagents used shows well that the ideal catalyst was not found yet for the isomerization of lactose to lactulose. This catalyst must answer some important criteria, among which are enumerated by the following: It must guarantee a maximum level of isomerization with a minimum of reaction by-products;
To be environmentally safe and not toxic; The cost of the catalyst must be as possible low and to be available in great quantity; It must be easy to remove from the medium by traditional demineralization tools; To give repetitive results of isomerization. However, in practice, the catalysts used for the isomerization of lactose to lactulose present positive and negative aspects. Systematic analysis of the most used catalysts for lactulose production could be divided into three principal groups. They are strong acids, strong bases and amphoteric catalysts represented mainly by hydroxyls, sulphites and borates. Principal methods as well as the reactions which control the process of isomerization of lactose into lactulose will be treated in what follows. Lactose isomerization by hydroxyls Lactulose was obtained for the first time by Montgomery and Hudson (1930) following the heating of a solution made up of a mixture of lactose and lime at a temperature of 35 S during several days. In order to obtain crystalline lactulose, several stages of purification were used. Reagents such as sulphuric acid, calcium carbonate, ethanol, methanol, ether, activated carbon and bromine were used. Thereafter, a new method was developed (Matvievsky, 1979; Russian Patent No 1392104, 1988; U.S. Patent No . 3272705, 1966; Yakovleva, 1963) in which calcium hydroxide was used as catalyst of the isomerization reaction of lactose to lactulose. In this method, 60% lactose solution was combined with 0.1% of calcium hydroxide under a temperature of 100e102 C and reaction time of 15 min. Thereafter, final solution was demineralised by combination of electrolysis and ion exchange resins. However, this method was rather tiresome. In the work reported in Patent of Germany No 222468033 (1976), the use of various catalysts such as (Ca(OH)2, NaOH, CaO, Na2CO3, KOH, K2HPO4, Ba (OH)2) was reported (Dalev & Tsoneva, 1982; Lodigin, 1999; Patent of Germany No 297999, 1992; Pereligin, Podgornov, & Sitnikov, 1999; Russian Patent No
Fig. 1. Schematic representation of lactulose molecule.
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Fig. 2. Representation of the Lobry de Bruynevan Ekenstein transformation.
2101358, 1998; Russian Patent No 97120603/13, 1999; Stomberg & Semchenko, 1999; U.S. Patent No 4264763, 1978). In this study, effect of these catalysts on galactose rate as by-product of disaccharides (lactose and/or lactulose) decomposition (degradation) was also reported. While being based on results reported in Patent of Germany No 222468033 (1976), the following conclusions could be mentioned: Independently on the quantity of NaOH used, there exists a linear dependence between galactose rate produced as reaction by-product and lactulose rate which represents rate the reaction isomerization rate. This relation seems to increase considerable at isomerization rates higher than 21.4%; Galactose/lactulose ratio remains constant for a constant ratio NaOH/lactose independently on isomerization time and temperature. Basing on results reported in Patent of Germany No 222468033 (1976), an industrial application was carried out (France Patent No 2147925, 1973; Patent of Germany No 222468033, 1976). In the process used for the industrial production, lactulose was purified and demineralised by anion and cation exchange resins. The end product had a concentration of the lactulose of about 42e48% of total dry matter. However, even if this method was effective, it remains that it needed rather important material and energy expenditure. Another method was developed in Russian patent No 7374626 (1980). In this method, 15e20% of lactose solution was mixed with 0.35e0.45% of the calcium hydroxide to reach pH 11. The mixture was thermostated
Fig. 3. Schematization of the Amadori rearrangement.
at 68e72 S during 15e20 min. The final solution pH was 8.8e9.0. Thereafter, solution was neutralized with citric acid up to pH 5.5e6.5. Citric acid quantity added in a form of saturated solution was 0.115e0.125%. The pH decrease was carried out to avoid an autocatalytic degradation of lactulose and to facilitate partial demineralization of final solution by means of calcium citrate formation (calcium complexation) removed by centrifugation. An additional operation of demineralization with of ions exchange resins was necessary. Effect of lactose concentration. According to fundamental laws of chemical kinetics, the lactose isomerization process into lactulose depends on lactose concentration in the feed solution. Systematic analysis of data on lactulose production showed significant variability of this parameter and it varies in the range of 5e60%. Moreover, the choice of lactose optimal concentration in the feed solution depends mainly on type of the catalyst. Using hydroxides (sodium, potassium or calcium) as catalysts and in order to determine the optimal lactose concentration giving maximum lactulose reaction rate with minimum coloration of the final solution, experiments with lactose concentrations varied between 5% and 30% were carried out (Ryabtsova, 1992). Lactose used in this work was of food grade but not refined. It was purified from proteins to avoid reaction between lactose and protein amine groups. Isomerization reaction was carried out by using sodium hydroxide as catalyst with an initial solution pH of 11.0 0.2 under 70 2 S and reaction time of 20 2 min. Data reported by Ryabtsova (1992) showed that lactose concentration in the range of 5e30% did not have any significant effect on the reaction isomerization rate. However, these same data showed that at lactose initial concentration higher than 20%, reaction by-products rate was higher then when lower lactose concentration were used. Same results were reported with calcium hydroxide as catalyst. Moreover, to avoid lactose crystallization after cooling the solution, it is important to use concentration in the range of 20e25%. In one other study (Montilla, del Castillo, Sanz, & Olano, 2005); concentrates of whole whey and ultrafiltrates were used to produce lactulose. Whole milk whey and milk
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whey ultrafiltrate concentrated up to 5.2 and 7.6 fold were used. Results indicated that the amount of produced lactulose increased by increasing the concentration factor of the raw material. Indeed, using simple milk, 0.388 and 1.17 g/100 ml were obtained after 30 and 120 min, respectively, of treatment. By concentrating the whey with a concentration factor up to 5.2 fold, the amounts of produced lactulose were 3.41 and 4.80 g/100 ml after 30 and 120 min, respectively. Further concentration of the initial milk whey up to a concentration factor of 7.6 fold permitted to obtain lactulose concentrations of 5.33 and 7.15 g/100 ml after 30 and 120 min of treatment (Montilla et al., 2005).
Effect of lactose and catalyst concentrations on solution pH. In the case of lactose isomerization into lactulose, solution pH characterizes concentration of proton acceptors and it plays a very important role and could be regarded as one of the more influencing factors on the isomerization reaction of lactose into lactulose. For this reason, combined effect of lactose concentration and alkaline catalyst on pH of the medium was studied by several authors. Also, this aimed to determine optimal amount of the catalyst to maintain solution pH in favourable interval for a maximum isomerization with a minimal rate of the reaction byproducts. Experimental results showed that there is no significant difference between pH values of model solutions composed by lactose of high purity compared with refined lactose solutions. Moreover, analysis of experimental data showed that independently on lactose concentration, curve of solution pH evolution shows logarithmic behavior. During first reaction stage, solution pH abruptly increases and it was reported that to increase pH of lactose solution from 5.5 0.5 up to 9.0 0.2, NaOH concentration needed is the same one independently on lactose concentration. In [100], NaOH concentration needed to increase pH from 5.5 0.5 up to 9.0 0.2 is 0.004 0.001 M. At the second stage of pH curve evolution, increase of pH was very low. During period, lactose concentration had significant effect on pH increase. It is more difficult to increase the pH of the concentrated lactose solutions. For example, to increase pH up to 11.0e11.5 of lactose solution of concentration of 0.015e0.03 M it was necessary to add 0.03e0.05 M of NaOH and for lactose solution of a concentration of 0.06 M, NaOH concentration of 0.06e0.07 M was needed. In dilute media, no significant difference was reported on NaOH concentration needed to increase pH from 5.5 0.5 up to 11.5 0.1 between raw and refined lactose. But, for lactose concentration above 0.06 M, to reach the same pH (11.5 0.1), it is necessary to use more NaOH in the case of raw lactose then with refined lactose. This difference is caused by the presence of minerals and nitrogenized compounds with high buffer capacity. Similar results as those obtained with NaOH were reported when Ca(OH)2 was used as catalyst of the isomerization reaction
of lactose into lactulose. To reach pH value of 11.0 0.2, concentrations of 0.02e0.04 and 0.05.0.06 M of Ca(OH)2 were added to 0.015e0.03 and 0.06 N lactose solutions, respectively (Ryabtsova, 1992). As general conclusion on relationship between lactose solution concentration and catalyst concentration, it could be resumed as follows: at pH 11.0 for each 1 M NaOH it needs 10 M of model solution lactose independently on concentration; 3e7 M NaOH in the case of concentrated lactose solution from milk whey dependently on the cheese process and finally 1e2 M NaOH for lactose solution made from raw lactose of high quality. So, we can see that by increasing lactose solution buffer capacity, lactose/catalyst ratio decreases. In general, it is important to know purity of lactose for a good choice of an optimal catalyst concentration because differences between results obtained with model lactose solutions and real solutions are significant (Ryabtsova, 1992). It was also reported that during the isomerization of lactose to lactulose with sodium hydroxide, a high level of degradation occurred and to decrease the amount of the formed reaction by-products, it is better to use the lower ratios of sodium hydroxide to lactose (about 0.5% w/w). In this case the maximum conversion of lactose to lactulose was about 20% and total by-product is about 5e7% (Zokaee et al., 2002).
Particularities of lactose isomerization by sodium and calcium hydroxide. Isomerization of lactose into lactulose with NaOH as catalyst of the reaction was carried out by using raw lactose solution of high quality at a concentration of 20%. Initial solution pH was fixed at 11.0 0.2. Lactose solution was thermostated in batch mode at 70 C. Samples were analyzed for optical density, pH evolution and isomerization rate which was represented by lactose/lactulose ratio. Experimental data reported by Ryabtsova (1992) showed that at the first stage of the isomerization reaction, there was a considerable growth of lactulose rate with a light change of solution color which characterizes formation of reaction by-products. The second reaction stage was characterized by a stability followed by a decrease of the isomerization rate. This was more intense when pure lactose solutions were used. During this stage, the decrease pH was significant and followed by an intensification of the solution color. The pH decrease was also intensified by the formation of reaction by-products with an acid character. Decrease of solution pH is an indication of the decease of proton acceptors concentration which in its turn diminishes the probability of intramolecular regrouping of lactose and its isomerization into lactulose. Moreover, because of pH decrease, lactulose degradation resulting in galactose and fructose formation could be accelerated. Another phenomenon is also possible following pH decrease which is the reversible isomerization of the lactose into lactulose through an enolic form. Same results were reported with calcium hydroxide as catalyst
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of lactose isomerization reaction. However, calcium hydroxide intensification of solution mixing is important to avoid precipitation of the catalyst. In general, it appears from systematic data analysis that maintenance of high alkalinity of the medium is an essential condition for an optimal isomerization rate of lactose into lactulose.
Formation of isomerization reaction by-products. Analysis of data on the variation of optical density and pH during degradation of sugars in alkaline media showed existence of three stages, which are closely related to the stages of lactulose formation during lactose isomerization. The first stage corresponds to a minimal degradation and this period corresponds to the highest level of lactulose formation. The second stage corresponds to the growth of sugars degradation by-products. This stage is followed by pH decrease and lactulose formation. Finally, a third stage which is characterized by a stabilization of solution color growth even at relatively high reaction temperature (72 2 C). During this third stage, pH decrease could reach neutrality. However, even if measurement of solution optical density could not give sufficient information about rate and nature of the formed dyes following lactose and/or lactulose degradation, it remains that this information could be used for better planning of lactulose production process, especially with regard to stages of residual lactose crystallization and lactulose refining which is the end product of the process (Rudenko, 1999; Rudenko & Bobrovnik, 1999). In order to better understand the process of color growth (dye formation) during isomerization of lactose into lactulose, 20% lactose solution was studied with sodium and calcium hydroxide as catalyst under temperature of 72 2 C. Results reported by Ryabtsova (1992) on optical density measurements showed that in the region of visible spectrum, growth of the absorbance was linear and that the maximum was reported to a wavelength of 360 nm. By increasing the reaction time, highly significant increase of the mixture optical density was recorded. This could be explained by the fact that in the zone of visible spectrum, different functional groups of dyes products have identical absorbance spectra which differ only by intensity as reported in Sapronov (1975). By increasing reaction time with sodium hydroxide as catalyst, a spectrum of absorbance to 490 nm was shifted and this could correspond to a change of the ratio between reaction by-products. The same data were reported using calcium hydroxide as catalyst but with a longer reaction time compared to NaOH. Optical density measurements of 0.5% lactose mixed with lactulose solution in UV zone showed maximal absorbance at 270e280 nm, which is a zone of the absorbance of lactose. Once the mixed solution of lactose/lactulose was thermostated under temperature of 72 2 C, the shape of the initial spectrum had changed significantly and other spectra of absorbance appeared. By increasing reaction
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time, maximum of absorbance was moved towards 260e 270 nm, which corresponds to the zone of absorbance of lactose/lactulose degradation by-products in alkaline media. Study of UV spectrum during lactose isomerization into lactulose could be used as base to confirm that the growth color in solution under reaction could be caused by degradation of reducing sugars when NaOH was used as catalysts (Parrish, Hicks, & Doner, 1980).
Isomerization of lactose by sulphites and phosphates Sulphites and phosphates have the characteristic to prevent oxidation of disaccharides and for this reason their use as catalyst of lactose isomerization reaction into lactulose allows the use of high temperatures and high lactose concentrations. According to data reported in Patent of Austria No 288595 (1971), for lactulose production, lactose solutions of 60e65% were used and temperature of 80e100 S. Under these conditions, sulphites were added at a rate of 0.05e0.05 M per kg of lactose monohydrate. Thereafter, the mixture (lactose solution with catalyst) was thermostated at this temperature until obtaining a constant value of the optical rotation of the solution. Then, the solution was cooled followed by crystallization of part of residual lactose. After crystallization, the solution of lactose/lactulose was treated by ion exchange resins for purification from sulphites (catalyst) and organic acids. Following this operation, another part of lactose was crystallized by cooling. To accelerate the crystallization process of lactose, it was recommended to add a sowing in the form of fine lactose crystals for a maximum crystallization yield (Polyansky & Shestov, 1995). The end product was syrup with 54.5% of dry matter, 38.7% of the lactulose. Galactose and lactose contents in the final product were about 8.2% and 3.8%, respectively. Another method for lactulose production was reported in Patent of Great Brittan No 2031430 (1980). According to the method described by the authors, 2.1e8.6% of phosphates was added to a saturated lactose solution. The temperature of the reaction was 104 S during 20e240 min, dependently of the ratio lactose/catalyst. In this case, maximum isomerization rate reported was 20%. At the end of reaction time, part of lactose was crystallized by cooling and was removed from the medium by filtration. The remainder solution was diluted up to 15% and treated by anion and cation exchange resins for purification from organic acids formed as reaction byproducts and phosphate (catalyst). After this operation, the solution was concentrated another time and part of lactose was removed by crystallization. Other methods were also reported (U.S. Patent No 4536221, 1985). The catalyst used in these cases was a mixture of sodium hydroxide and sodium sulphite at a concentration of 0.3e1% with 60% lactose solution under a temperature of 75e80 S during 15 min. The isomerization rate reported reached 30%. At the other hand, using 0.7% of sodium sulphite as catalyst, isomerization rate reached 40%. In the patent described
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in U.S. Patent No 4536221 (1985), the use of magnesium hydroxide mixed with sodium hydrosulphide in lactose solution of 60e70% concentration was reported. The catalyst concentration in this case was 0.05e0.2% and the temperature used was 90e100 S.
Isomerization of lactose by aluminates and borates Using amphoteric electrolytes such aluminium hydroxide or boric acid, total isomerization yield of lactose into lactulose could reach 70e80% (European Patent 0320670, 1990; Mendicino, 1960; U.S. Patent 4273922, 1981). In these cases, the catalyst must be added at a rate of 0.5e4 M per mole of lactose as reported in the US patent No 4957564. In U.S. Patent No 3546206 (1970), it was reported that catalyst (aluminate) was added to lactose solution. Thereafter, the mixture was heated and thermostated a certain time and then the catalyst was removed from the medium using crystallization by cooling. The pH was readjusted with HCl or aluminium hydroxide, dependently on the case. At the end of the process, it was recommended to isolate lactulose using methanol. Carrobi and Innocenti (1990) proposed using of membrane to remove catalyst after isomerization. Following this work, patent was deposited (U.S. Patent No 4957564, 1990). In these patents, it was reported that 25e50% lactose solution was used. The catalyst (sodium aluminate) was added in the form of 35e45% concentration solution. The ratio aluminate/lactose was 0.3/1 up to 1/1, dependently on lactose concentration. Isomerization was carried out under a temperature of 50e70 C during 30e60 min. At the end of the reaction time, solution was neutralized with 3e4 N sulphuric acid to keep pH in the range of 4.5e8.0. Aluminium hydroxide suspension was formed and then removed from the medium by centrifugation followed by membrane treatment. Other authors reported the use of sodium tetraborate, sodium hydroxide or triethylamine mixed with boric acid as catalyst (Hicks, Raupp, & Smith, 1984; Mizota et al., 1987). Crystallization, pasteurization and purification operations by ion exchange resins were necessary.
Isomerization of lactose by alkaline-substituted sepiolites De la Fuente, Jua´rez, de Rafael, Villamiel, and Olano (1999) reported that strong base catalysts, prepared by substituting a part of the Mg2þ located at the borders of the channels of sepiolite with alkaline ions (Liþ, Naþ, Kþ and Csþ), were investigated as catalysts for the isomerization of lactose to lactulose and epilactose. The activities exhibited by alkaline-exchanged sepiolites were significantly higher than that of natural sepiolite. The influence of temperature, time of the reaction and catalyst loading were also evaluated. A 20% conversion was obtained at 90 C at a catalyst loading of 15 g/l. At the other hand, Villamiel, Corzo, Foda, Montes, and Olano (2002) reported
that alkaline-substituted (Naþ, Kþ) sepiolites were used as catalysts for the formation of lactulose in milk permeate. Besides lactose and lactulose, other carbohydrates, such as galactose and epilactose, produced in side reactions, were determined. The effect of different washing cycles of sepiolite on the isomerization of lactose and the exchange of cations with the permeate was also investigated. In general, the activity of the sodium sepiolite was higher than that of potassium form. Twenty per cent of lactulose formation (1000 mg/100 ml), with respect to the initial lactose, was obtained after 150 min of reaction, using sodium sepiolite washed during 10 cycles. Under these conditions, 25% of lactose degradation was detected, whereas small amounts of epilactose and galactose were formed. The exchange of Naþ between sepiolite and permeate decreased considerably with the number of washing cycles. The present work shows an appropriate method for obtaining lactulose in milk permeate with acceptable yields and without complicated purification steps. Lactulose production by ion exchange resins Production of lactulose in a form of concentrated solutions or powder for use as additive in functional food is more and more of topicality. This type of product must be at the same time safe and easy to produce, whether for environment or human consumption. Based on this concept, several works were carried out during the 10 last years in order to find effective technologies for lactose isomerization into lactulose without use of reactive agents. As it was mentioned above, lactulose production requires use of chemicals (hydroxide, borates, aluminates, etc.) and purification procedures by ion exchange resins. In the Russian Patent No 2101358 (1998), anion exchange resins were used to intensify process isomerization of lactose into lactulose by exploiting OH ions exchange between solution in reaction and resins and to simplify lactulose production process by using the same resins for demineralization of the end product. The advantages of this process are There is no need to add catalyst for isomerization process; Demineralization stage is not used; No additional operation to purify the end product from dyes; He process is more profitable in comparison with traditional methods; Lactulose produces by this technology could be used in functional food for children, and in specialized food into which bifidobacteria are introduced (Hramtsov et al., 2004). Concluding remarks Lactulose production is a complex process, which is affected by several operation and technological conditions.
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Most of the changes incurred to the lactulose by heating are disadvantageous to quality of the final product. However, degradations can be minimized by appropriate design of the isomerization process, type of catalyst used and lactose quality. Designing process of lactose isomerization into lactulose must be done in a comprehensive way considering pre-isomerization, isomerization and postisomerization processes. Isomerization of lactose into lactulose must be preceded by adequately chosen raw material yields with expected quality and optimal lactose/ catalyst ratio. That quality can be maintained during lactulose isolation by application of appropriate post-processing parameters. Because of complex influence on the product and many technological variables, which can be controlled during processing, isomerization is a versatile way to treatlactose, which is an abundant product of cheese processing. A thorough knowledge of pros and cons of the isomerization process is needed in order to design optimal technological process for lactose isomerization into lactulose with a minimum of by-products and to obtain final product of desired quality for a variety of use.
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