The bacteria were identified with FISH. Twenty-eight healthy Chinese subjects temporarily living in The Netherlands, 16 female and 12 male, age range 20—31 years were recruited for this study. All subjects did not have diabetes or gastrointestinal disorders and none had taken antibiotics or laxatives during the three months prior to the study.
Every subject signed a declaration of informed consent. All faecal samples were processed within 2 h of defecation. Samples were homogenized by mechanical kneading for 2 min. For each sample, 10 g was suspended in 40 ml of an anaerobic salt solution pH: 7. After incubation for 72 h, for each faecal sample, the dilution with 20— colonies per plate was selected for enumeration of the total amount of colonies.
The colonies on the plate were transferred to a Whatman filter Qualitative, Whatman International Ltd, Maidstone, England , during which both the filter and agar were marked to facilitate orientation.
The filter with colonies was submerged in liquid nitrogen for 10 s to disrupt the cells. Separate colonies of diverse morphology were picked. Identification of bacteria was carried out using 16S rRNA oligonucleotide probes targeting the major bacterial groups in faeces. The probes are listed in Table 1 and were described earlier [ 7 ] with the exception of the Pep probe which was newly developed and validated in our laboratory data not shown.
Hybridization and visualization of fluorescent cells were carried out according to the methods described previously [ 8 ]. All probes have been described and validated elsewhere [ 7 ] except for Pep which was recently validated in our laboratory unpublished. After donating faecal samples, the 28 subjects first underwent a challenge of 25 g glucose and a challenge of 25 g of lactose in two blind tests.
SSC was recorded for 6 h after the challenge according to the method described earlier [ 2 ]. The purpose of the glucose and lactose challenges was to select subjects who did not report complaints after glucose ingestion. Among the 28 subjects, 20 reported a SSC of less than 2 after glucose and they underwent another challenge of 25 g of lactose, after which SSC was also recorded.
Of the 28 subjects, 12 were defined as lactose tolerant or intolerant according to the criteria mentioned above. Correlation was assessed by calculating the Spearman correlation coefficients. All analyses were performed using SPSS For the 28 samples, the counts of total bacteria cultivated on the BBA agar were 0. On average, the enzyme was found to be present in The average and SD of the percentages of blue colonies responding to ato, chislit, elg01c, lab, pep, rbrorfla and strc probes were not calculated because they were only detected in a few samples.
In total, of the blue colonies were chosen randomly for identification with FISH, of which were identified with the 14 group-specific probes. No colony responded positively to the probes for Enterobacteriaceae, Eubacterium cylindroides group, Phascolarctobacterium group or Veillonella , therefore these probes are not included in Table 2.
The other 16 subjects could not be classified into either of the two groups according to our criteria. Favier et al. Faecal samples were cultured on an enriched Columbia agar medium modified by addition of X-gal. Some of these colonies were sub-cultured for identification by classical biochemical and morphological characteristics.
It was assumed that the modified media in some way favoured the growth of Bifidobacterium , which did not reflect the true conditions in the faeces. When the method was applied to the faecal samples from 28 subjects with genetically determined low lactase activity, on average This is higher than reported by Favier et al. Results in this study indicate that a major part of the colonic microbiota is capable of hydrolyzing lactose, as described earlier [ 6 ]. In the study by Favier et al.
In this study, 7 other groups besides the three groups, i. For the last few years, the FISH technique has been employed to identify and quantify colonic microbiota [ 7 , 14 ]. During method development, 60 white colonies were also picked from cultures of three faecal samples on Columbia, Wilkins-Chalgren or BBA agar with X-gal.
There has been discussion of the advantages and disadvantages of using faeces to study the composition and activities of the colonic microbiota. Some studies found that the bacteria in faeces reflect the microbiota in the colon [ 15 ] while others studies did not [ 16 ]. The metabolic activities measured in faeces can be quite different to those in the proximal colon, where there is active bacterial fermentation of carbohydrates. Faecal samples provide a reasonable alternative for comparing lactose-fermenting capacities and composition of the colonic microbiota among lactose maldigesters, particularly when considering difficulties in sampling in the colon.
Some studies found that carbohydrate fermentation properties of faecal bacteria are individual-dependent and rather stable through time [ 17 , 18 ]. Considering the fact that lactose maldigestion correlates poorly with the symptoms of intolerance and the possible bias in scoring symptoms through familiarization with the test procedures or placebo effect [ 19 ], a placebo control glucose was included besides two lactose challenges in an attempt to reliably define groups of lactose tolerance and intolerance.
Based on the observation that a major part of the faecal microbiota is capable of hydrolyzing lactose, it is unlikely that lactose itself will present a large osmotic threat in the colon, as it should be quickly degraded by the majority of colonic microbiota.
It is not yet known whether the subsequent processes of bacterial fermentation after the hydrolysis of lactose might play a role in lactose intolerance. Furthermore, besides the fermentation process of lactose, other colonic factors, such as the colonic capacity to remove fermentation products, might also be related to lactose intolerance.
Large individual differences were also observed in the numbers of total bacteria and main groups of bacteria in faeces in a previous study [ 5 ]. The possible explanation for this could be that enzyme activity may vary considerably among lactose-fermenting bacteria.
The total enzyme activity in faeces is determined not only by the amount but also by enzyme activity of the bacteria with the enzyme. The method was applied to investigate faecal microbiota of subjects with genetically determined low lactase activity.
We thank Gerwin C. Raangs, Sandra P. Harmsen for inspiring discussions. We also thank the subjects who kindly participated in our study. Vesa T. Marteau P. Korpela R. Google Scholar. Vonk R. Priebe M. Koetse H. Although the role elastin-binding protein plays in the development of GM1 gangliosidosis is unclear, the alteration of this protein may contribute to the weakened heart muscle cardiomyopathy found in some people with GM1 gangliosidosis. Most of these mutations change single nucleotides in the gene.
The degradation of GM1 ganglioside is not affected by these mutations. Because keratan sulfate is predominantly found in cartilage and the cornea, the buildup of this substance causes skeletal abnormalities and cloudy corneas. Researchers believe that a buildup of GAGs may also cause the features of MPS IV by interfering with the functions of other proteins inside lysosomes and disrupting the movement of molecules inside the cell.
Genetics Home Reference has merged with MedlinePlus. Learn more. The information on this site should not be used as a substitute for professional medical care or advice. Contact a health care provider if you have questions about your health. GLB1 gene galactosidase beta 1. From Genetics Home Reference. Furthermore, lactose is a hygroscopic sugar and has a strong tendency to absorb flavours and odours and causes many defects in refrigerated foods such as crystallization in dairy foods, development of sandy or gritty texture, and deposit formation [ 2 ].
Technologically, lactose gets easily crystallized, which sets the limits of its applications to certain processes in the dairy industry. Cheese manufactured from hydrolyzed milk ripens more quickly than that made from normal milk. Treatment of milk and milk products with lactase to reduce their lactose content seems to be an appropriate method to increase their potential uses and to deal with the problems of lactose insolubility and lack of sweetness.
Furthermore, this treatment could make milk, a most suitable food, available to a large number of adults and children that are lactose intolerant. Moreover, the hydrolysis of whey converts lactose into a very useful product like sweet syrup, which can be used in various processes of dairy, confectionary, baking, and soft drink industries [ 3 , 4 ].
Therefore, lactose hydrolysis not only allows the milk consumption by lactose intolerant population but can also solve the environmental problems linked with whey disposal [ 5 — 7 ]. These were widely recognized as the nondigestible oligosaccharides, not hydrolyzed or absorbed in the upper intestinal tract, and they pass onto the colon where they are fermented selectively by beneficial intestinal bacteria.
Besides their prebiotic effects, these GOSs have low cariogenicity, low caloric values, and low sweetness [ 8 , 9 ]. GOSs occur naturally in trace amounts in breast milk, cow milk, honey, and a variety of fruits and vegetables [ 10 ]. As a result, increased production of GOS is useful. The use of immobilization technology is of significant importance from economic point of view since it makes reutilization of the enzyme and continuous operation possible and also precludes the need to separate the cells from the whey following processing.
It can also help to improve the enzyme stability. Microorganisms offer various advantages over other available sources such as easy handling, higher multiplication rate, and high production yield.
However, they differ in their optimum conditions for the enzyme application especially pH range. A recovery cost of the enzyme depends on the level of production and purification.
Therefore, there has been increasing interest in finding microorganisms with adequate properties for industrial use, higher production capacity, and less expensive purification methods of this enzyme. Thermoanaerobacter has been purified by chromatography through DEAE-cellulose [ 27 ]. Lactobacillus delbrueckii subsp. Lysozyme enzyme treatment was determined as the most effective method, which resulted in approximately 1. Both enzymes showed a remarkable hydrolytic activity and a weak transgalactosilation activity, even in the presence of high concentrations of lactose.
The thermophilic enzyme showed a higher resistance to hydrophobic agents and a higher stability at different temperatures, pHs, and chemical conditions. However, the enzyme of Thermus was less stable in the presence of oxygen peroxide, showing that some residues important for its stability were affected by oxidation.
The enzyme from K. The thermophilic enzyme was competitively inhibited by galactose much strongly than its mesophilic counterpart but the inhibition did not change with the temperature.
To improve the enzyme purification a selective one-point adsorption was achieved by designing tailor-made low-activated Co-iminodiacetic acid Co-IDA or Ni-IDA supports. The new enzyme was not only useful for industrial purposes but also has become an excellent model to study the purification of large multimeric proteins via selective adsorption on tailor-made immobilized metal-ion affinity chromatography supports.
Strain T2 has been purified and immobilized in a single step, combining the excellent properties of epoxy groups for enzyme immobilization with the good performance of immobilized metal-chelate affinity chromatography for protein purification [ 36 ]. The optimum pH range for the fungal enzyme is 2. The enzyme was purified 6. With neutral pH optima, these are well suited for hydrolysis of lactose in milk and are widely accepted as safe for use in foods.
Further, the partial purification of the enzyme from K. The studies on the use of fed batch culture techniques to achieve high culture productivity in K. The optimized condition for the enzyme production was reported as follows: temperature Thus, there is a need to explore their full potential as catalyst by adopting suitable strategies for enzyme stabilization.
The multimeric enzyme can be stabilized by using proper experimental conditions and genetic tools to cross link or to strengthen the subunit-subunit interaction [ 49 ]. The stability of monomeric or multimeric enzymes can also be enhanced by multipoint and multi-subunit covalent immobilization and enzyme engineering via immobilization [ 50 ].
The enzyme has been immobilized by various methods such as physical absorption, entrapment, and covalent binding method [ 51 — 85 ] on different supports Table 2. Physical adsorption is considered as the simplest method of immobilization in which an enzyme is immobilized onto a water-insoluble carrier and the biocatalysts are held on the surface of the carriers by physical forces van der waals forces. Frequently, however, additional forces are involved in the interaction between carrier and biocatalyst principally hydrophobic interactions, hydrogen bridges, and heteropolar ionic bonds [ 86 ].
This method has the advantage of being simple to carry out and has little influence on the conformation of the biocatalyst. However, the disadvantage of this technique is the relative weakness of the adsorptive binding forces. Different inorganic alumina, silica, porous glass, ceramics, diatomaceous earth, clay, bentonite, etc. Further adsorption of enzyme may be stabilized by glutaraldehyde treatment.
Enzyme activity of immobilized enzyme from K. When adsorption method was used, the highest activity was obtained with yeast enzyme and support Ostsorb-DEAE. The enzyme from A. Further, maximum immobilization occurred at pH 5. In particular, when the particle sizes of the support are increased, the enzymatic activity strongly decreases. However, the adsorption strength was much higher in the case of PEI-Sepabeads [ 53 ].
A recombinant thermostable B. The immobilization efficiency of Entrapment method is the physical enclosure of enzymes in a small space. Matrix and membrane entrapment including microcapsulation are the major methods of entrapment. The major advantage of the entrapment technique is the simplicity by which spherical particles can be obtained by dripping a polymer-cell suspension into a medium containing positively charged ions or through thermal polymerization [ 86 ].
Further, beads formed particularly from alginate are transparent and generally mechanically stable. The major limitation of this technique for the immobilization of enzymes is the possible slow leakage during continuous use in view of the small molecular size compared to the cells.
However, improvements can be made by using suitable linking procedures. The matrices used for the immobilization are usually made up of polymeric materials such as Ca-alginate, agar, k- carragenin, polyacrylamide, and collagen. However, some solid matrices such as activated carbon, porous ceramic, and diatomaceous earth can also be used for the immobilization. The membranes commonly used for the entrapment of enzymes are nylon, cellulose, polysulfone, and polyacrylmide. The glutaraldehyde-treated K.
The alginate beads obtained after treatment with polyethyleneimine followed by glutaraldehyde solution were stable. The entrapment of A. Moreover, the optimum conditions were also not affected by immobilization [ 95 ]. Covalent binding is the retention of enzymes on support surface by covalent bond formation. Enzyme molecules bind to support material via certain functional groups such as amino, carboxyl, hydroxyl, and sulfydryl groups.
These functional groups must not be in the active site. It is often advisable to carry out the immobilization in the presence of its substrate or a competitive inhibitor so as to protect the active site Functional groups on support material usually activated by using chemical reagents such as cyanogen bromide, carbodimide, and glutaraldehyde.
Fungal enzyme from A. Zeolites were nonideal since its coupling yield was low whereas nylon resulted in a stable matrix. The derivatives obtained either by diazo or by carbodiimide coupling showed the highest activities during immobilization of the enzyme on glycophase-coated porous glass [ ].
The enzyme immobilized on silica-alumina was more stable than the free form at acidic pH [ 65 ]. The heat stability of lactase can be increased through immobilization [ 66 , , ]. The free lactase has been cross-linked into Fe 3 O 4 -chitosan magnetic microspheres for lactulose synthesis by dual-enzymatic method in organic-aqueous two-phase media using lactose and fructose as the raw materials [ ].
The organic-aqueous media can significantly promote the transglycosidation activity of lactase and therefore improves the lactulose yield. Immobilization of the enzyme on heterofunctional epoxy Sepabeads boronate-epoxy-Sepabeads and chelate-epoxy-Sepabeads has shown considerable results in reducing the product inhibition [ 55 ].
The effect of internal mass transfer and product galactose inhibition on a simulated immobilized enzyme-catalyzed reactor for lactose hydrolysis has been studied [ ].
A general mathematical model has been developed for predicting the performance and simulation of a packed-bed immobilized enzyme reactor performing lactose hydrolysis, which follows Michaelis-Menten kinetics with competitive product galactose inhibition. The performance characteristics of a packed-bed-immobilized enzyme reactor have been analyzed taking into account the effects of various diffusional phenomena like axial dispersion and internal and external mass transfer limitations.
The effects of intraparticle diffusion resistances, external mass transfer, and axial dispersion have been studied and their effects were shown to reduce internal effectiveness factor.
The choice of process technology depends on the nature of the substrate, the characteristics of the enzyme, economics of production, and marketing of the product. The primary characteristic, which determines the choice and application of a given enzyme, is the operational pH range.
Acid-pH enzymes from fungi are suitable for processing of acid whey and whey permeate whereas the neutral-pH enzymes from yeasts and bacteria are suitable for processing of milk and sweet whey. Lactose-hydrolyzed milk has been used for the preparation of flavoured milk, cheese, and yoghurt. The hydrolysis of lactose in milk for food processing also prevents lactose crystallization in frozen and condensed milk products.
Moreover, the use of hydrolyzed milk in yoghurt and cheese manufacture accelerates the acidification process, because lactose hydrolysis is normally the rate-limiting step of the process, which reduces the set time of yoghurt and accelerates the development of structure and flavour in cheese [ 1 ].
The quality of ice milk and ice-cream was significantly improved by addition of lactozyme. It prevents the crystallization of lactose by breaking into glucose and galactose and reduces sandiness [ ].
High concentration of lactose in whey is a major environmental problem since its disposal in local water streams increases the biological oxygen demand manifolds.
Concentrated hydrolyzed whey or whey permeates can be used as a sweetener in products such as canned fruit syrups and soft drinks [ 1 ]. Further, the operational stability was tested, with the system being used up to 5 times before any significant drop in the activity.
The immobilized K. It has been observed that lactose conversion decreased the stability of milk casein particles and increased its dispersity [ ]. It was observed that specific activity yield decreased with the increase of the enzyme loading [ ].
Both the free and the immobilized enzymes are competitively inhibited by galactose, while glucose inhibited only the action of free enzyme, in an uncompetitive way. The immobilization step helped to eliminate the inhibition by glucose. Moreover, the immobilization reduced to a half the inhibitory action of galactose. In general, the immobilization reduced the activity of the enzyme but increased its thermal stability.
Among the different treatments microfiltration, thermal treatment, and ultrafiltration of whey, ultrafiltration was the best treatment towards a proper substrate solution for feeding the reactor [ 84 ]. Recently, a packed bed reactor together with alginate entrapped permeabilized cells K. Moreover, the transferase reaction can be used to attach galactose to other chemicals, resulting in formation of galacto-oligosaccharides GOSs , and consequently have potential application in the production of food ingredients, pharmaceuticals, and other biological active compounds.
Oligosaccharides are recognized as useful dietary tools for the modulation of the colonic microflora toward a healthy balance. This usually involves selectively increasing the levels of gut Bifidobacteria and Lactobacilli at the expense of less-desirable organisms such as Escherichia coli , Clostridia , and proteolytic bacteroides [ ].
The amount and nature of oligosaccharides formed depend upon the several factors including the enzyme source, the concentration and nature of the substrate, and reaction conditions [ 12 , , ]. The yield of oligosaccharides can be increased by using higher substrate concentrations or decreasing the water content [ 31 ].
The reaction conditions for transgalactosylation should be high lactose concentration, elevated temperature, and low water activity in the reaction medium [ ]. The temperature, concentration of substrate, and enzyme origin play an important role in the enzymatic synthesis of oligosaccharides [ ].
However, the influence of the initial lactose concentration can be much larger [ , ]. In general, more and larger galacto-oligosaccharides GOSs can be produced with higher initial lactose concentrations. The higher temperatures can be beneficial in higher oligosaccharide yields.
The higher yield at higher temperatures is an additional advantage when operating at high initial lactose concentrations and consequently elevated temperatures. The enzyme immobilized on tosylate cotton cloth was used in plug-flow reactor for continuous production of galacto-oligosaccharide from lactose.
In general, more and larger GOS can be produced with higher initial lactose concentrations. The chitosan-immobilized A. An immobilized-enzyme system using polyethyleneimine, glutaraldehyde, and cotton cloth was studied and compared the galacto-oligosaccharide production in free-enzyme ultrafiltration and in immobilized-enzyme systems [ ]. The synthesis of galacto-oligosaccharides was optimized with respect to lactose concentration and enzyme to substrate ratio using immobilized A.
The immobilized A. The synthesis of galacto-oligosaccharides GOSs using A. GOS formation was not considerably affected by pH and temperature.
The concentrations of glucose and galactose encountered near maximum GOS concentration greatly inhibited the reactions and reduced GOS yield. Since the free enzyme cannot be reused, thus the resulting operation is not cost effective. After the desired lactose hydrolysis is achieved, cream is added to the hydrolysed milk to adjust its fat content. Although numerous hydrolysis systems have been investigated, only few of them have been scaled up and even fewer have been applied at an industrial or semi-industrial level.
The first company for the commercial hydrolysis of lactose in milk by using immobilized lactase was Centrale del Latte of Milan, Italy, utilizing the SNAM Progetti technology.
The process used an immobilized Saccharomyces Kluyveromyces lactis lactase entrapped in cellulose triacetate fibres. This reactor can also overcome the problem of channeling or severe pressure drop.
However, major problems associated with the immobilized enzyme system are microbial contamination, protein adherence, and channeling. Therefore, for long-term operations using immobilized system, periodic washing, and pasteurization are indispensable processes [ — ].
In immobilized enzyme system, protein adhered to the enzyme can be easily dissolved by using high and low pH solutions, because the immobilized enzyme has high durability over a wide range of pH. The immobilized enzyme can be pasteurized with benzalkonium chloride quaternary ammonium salt after removing the proteins.
The use of acetic acid solution as a cleaning and pasteurizing agent instead of lactic acid can also be effective. The problem of channeling observed in the packed column system can be overcome by changing the flow direction of feed during the operation [ , , ]. Therefore, the public demand for their production is significantly increased together with the development of an effective and inexpensive GOS production.
Major companies dealing with oligosaccharides production including GOS are in Japan [ ]. Recently, there is also an increasing trend of GOS production in Europe. Besides lactulose and soybean oligosaccharides, all oligosaccharides are prepared by transglycosylation from mono and disaccharides or by controlled hydrolysis of polysaccharides [ ]. Enzyme immobilization provides enzyme reutilization and may result in increased activity by providing a more suitable microenvironment for the enzyme.
Moreover, immobilized systems can provide better enzyme thermostability and pH tolerance. The periodic washing and pasteurization and flow direction of feed can solve these problems to great extent. The problem of microbial contamination can also be solved by exploiting the temperature property of the enzyme. On the other side, thermostable enzymes have the unique ability to retain their activity at higher temperatures for prolonged periods, and the process is less prone to microbial contamination due to higher operating temperature.
Thus, cold active and thermostable enzymes will have the great potential in the lactose hydrolysis and of particular interest to the researchers. Thus, immobilization enzyme systems will certainly find greater role in future times for the hydrolysis of milk, whey, and synthesis of galacto-oligosaccharides. Panesar et al. This is an open access article distributed under the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Article of the Year Award: Outstanding research contributions of , as selected by our Chief Editors. Read the winning articles. Special Issues. Academic Editor: Cristina M. Received 16 Jun Revised 22 Sep Accepted 21 Nov Published 27 Dec Source Microorganism s Bacteria Alicyclobacillus acidocaldarius subsp.
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