Chapter

Enzymes Applications for the Dairy Industry

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Abstract

Milk contains a number of indigenous enzymes that play an important role in processing and quality of milk for direct consumption and cheese. This chapter discusses only the use of exogenous enzymes, with particular emphasis to innovative applications. To this aim, applications have been classified into three main groups: use in cheese making, applications for shelf-life extension, and applications for functional and environmental purposes. Milk coagulants belong to four main categories: animal rennet, plant-derived coagulants, microbial coagulants, and genetically engineered chymosin. The principal application of exogenous lipases in the dairy industry is enhancing cheese flavor and acceleration of cheese ripening throughout controlled hydrolysis of triglycerides. The use of antimicrobial enzymes is a natural means to control microbial contamination, improving safety and shelf life of dairy products. Most applications involve lysozyme and lactoperoxidase. Transglutaminase (TG) has recently become of great interest to food scientists for its ability in strengthening the structure of protein gels.

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... Some examples of plant-derived protease include ficin from the fig tree, cinarase and cardosin from cardoon, bromelain from pineapple, and papain from papaya. These plants are a good source of coagulants for vegetarian cheese foods (Trani et al., 2017). However, the cheese produced using plant proteases lacks the correct flavor and texture compared to that obtained from animal origin enzymes. ...
... Microbial sources gained attention in the late 1980s and have been the most popular rennet source due to advancing recombinant technology and genetic engineering approaches (Law, 2009b;Pereira and Gimenez, 2016). Microbial sources of rennet production cost less due to their easy production through fermentation (Trani et al., 2017). Today around 50% of the world's cheese is produced using rennet from microbial sources. ...
... Lipases are obtained from animal sources such as epiglottis of calves, sheep, and goats (Trani et al., 2017). Commercial lipase is also produced from microbial sources such as Aspergillus, Mucor, Rhizopus, Candida, Pseudomonas, Achromobacter, Staphylococcus, and Bacillus species (Al-Manhel, 2018). ...
Chapter
Enzymes have found a great place in various industrial applications since the nineteenth century. The dairy industry has been the traditional user of enzymes for a wide range of applications, such as processing cheddar cheese, yogurt, milk, and milk products. There are various endogenous (present in milk) and exogenous enzymes (commercially produced) that play an essential role in dairy processing. The commercial enzymes (Exogenous) are added during various dairy processing steps for milk preservation and to improve milk quality. The global market for dairy enzyme production is quite large. However, it is mostly dominated by a few enzymes that include Rennet-Chymosin, Lipases, Protease, and B-galactosidase, which will be discussed in this chapter in detail, giving information about the properties, source, and production of these enzymes. Most of these exogenous enzymes are produced from microbial, plant, and animal sources. Additionally, with emerging technology and large-scale usage, genetic engineering has also been utilized to produce these enzymes through recombinant technology. `The production of proteinase, lactase, lipase, and microbial rennet is expanding in the research centers and small-scale operations because of their advantages. In not so distant future, the need for these enzyme proteins will be one of the most demanding ingredients because of the increasing importance of advanced healthy dairy products to curb malnourishment and obesity moving towards low-fat and healthy food products.
... TG catalyzes the posttranslational modification of proteins by transamidation of available glutamine residues by the formation of covalent cross-links between glutamine and lysine residues. Addition of transglutaminase to milk induces of cross-linking of caseins and whey proteins that improves the strength of milk gels (Trani, 2017: 170). Rennet cheese with modified textural and nutritional properties and improved yield could be obtained upon transglutaminase modification but simultaneous addition of rennet and transglutaminase is recommended. ...
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This study evaluated the activity of lactoferrin on the multiplication of probiotic Lactobacillus casei in vitro and in Minas fresh cheese. Growth curves of L. casei in BHI broth containing 1 mg/mL and 2 mg/mL of lactoferrin were performed. Additionally, Minas fresh cheeses added of L. casei (control) and added of L. casei and lactoferrin (2 mg/g and 4 mg/g) were produced and stored at 5°C during 28 days. Cheeses were analyzed for pH, titratable acidity and enumeration of L. casei and psychotropic microorganisms in days 1, 7, 14, 21 and 28 and for the centesimal composition in day 1. The experiment was repeated three times, and data were analyzed by ANOVA and Tukey test at 5% significance level. When tested in vitro, L. casei multiplication was stimulated by lactoferrin at a concentration of 2 mg/ mL, but this activity has not been verified in the cheese, even in that added by lactoferrin at 4 mg/g. Psychotropic population in the cheeses added by lactoferrin did not differ from control cheese (P>0.05), demonstrating that no antimicrobial activity occurred in the products. More studies should be performed to evaluate the antimicrobial effects of lactoferrin in foods, since they have more variables that may affect the activity of this protein when comparing to in vitro tests.
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A study was undertaken on Cacioricotta, a traditional Italian goat’s cheese obtained from overheated milk (90 °C) without use of starter. The profile of proteolysis in the artisanal type, made with vegetable coagulant (latex released from caprifig branches) as milk clotting agent, was compared to that of the “industrial” one, manufactured with calf rennet. Particular aim of the investigation was to study the differences and, possibly, establish a useful tool for distinguishing the two types of cheese. The study was based on the quantification of the water soluble, 15% TCA soluble and amino acid nitrogen fractions, RP-HPLC separation of low molecular weight peptides and their identification by mass spectrometry (MALDI-ToF MS). The use of fig latex was associated to higher amounts of the nitrogen fractions and to RP-HPLC chromatograms very rich in peptides, in contrast to an almost complete lack of peptides in the industrial counterpart. These results confirm the strong proteolyitic activity exerted by the caprifig clotting enzymes in spite of the intense overheating of the milk, which is considered to cause reduction of the rate of casein degradation in cheese. The MS-based identification of several peptides provided a support at the molecular level for the characterization of Cacioricotta made with this vegetable coagulant and could be useful for “tracing back” purposes. In conclusion, the peptide pattern determined by the use of caprifig for milk coagulation can be considered a particular feature of the artisanal Cacioricotta, giving confirmation of its vocation to EU protection as typical product.
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The natural antimicrobial system present in milk, the lactoperoxidase (LP) system, has been used to preserve raw milk quality in areas where it is not possible to use mechanical refrigeration for technical and/or economic reasons. Although the most widely recommended industrial application of the LP system in food production is in the dairy industry for the preservation of raw milk during storage and/or transportation to processing plants, other novel applications of the LP system are being explored. This review paper addresses the use of the LP system in the dairy industry, new research findings and developments made so far and the potential applications of the LP system in other food systems and commercial products. The paper also highlights the research gap and possible future research directions that deserve attention.
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A hygienized rennet paste (HRP) and a defined strain starter culture, including Lactobacillus casei subsp. casei IFPL as adjunct, were considered for manufacturing Majorero cheese, a Spanish traditional variety made from goat milk. Influence of both factors on physicochemical characteristics, proteolysis, rheological and sensory properties, was evaluated throughout the ripening. Cheeses produced either industrially (IL) or in artisanal manner (AL) were compared with the experimental lot (EL), which included HRP and IFPL starter in its manufacture. Results showed a low level of primary proteolysis, expressed by a low content of non-casein nitrogen (NCN), in experimental cheeses. Despite the slightly poor texture (hard and crumbly) related to the high TS and salt contents, a good general acceptability was attained for EL, with the best scores for aroma and flavour intensities achieved at 30 ripening days. In fact, the sensory panel detected the “piquant” flavour (typical of the artisanal cheese variety) in EL after 15 days of ripening.
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Lipases are a class of enzymes which catalyse the hydrolysis of long chain triglycerides. Microbial lipases are currently receiving much attention with the rapid development of enzyme technology. Lipases constitute the most important group of biocatalysts for biotechnological applications. This review describes various industrial applications of microbial lipases in the detergent, food, flavour industry, biocatalytic resolution of pharmaceuticals, esters and amino acid derivatives, making of fine chemicals, agrochemicals, use as biosensor, bioremediation and cosmetics and perfumery.
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The aim of anaerobic digestion of primary sewage sludge is to convert the carbonaceous material contained in the solids into methane and carbon dioxide. The products of digestion are therefore gases, stabilised sludge solids which are subsequently dewatered and disposed of, and sludge liquor which is generally further treated. This investigation assessed the impact of addition of hydrolytic enzymes to anaerobic digesters. Cellulase and pronase E were added singly and in combination, and it was found that the mixture of the two enzymes resulted in an 80% reduction in solids (cf. 20% in the control), 93% removal of particulate chemical oxygen demand (COD) (59% in the control) and 97% total COD removal (vs. 63%). The total suspended solids (TSS) concentration was reduced by 80%, from 25 g/l to 5 g/l. Single enzymes had little or no impact on sludge solubilisation, and final COD and TSS, but all of the enzyme additions were seen to decrease the production of volatile fatty acids (VFAs). Since accumulation of VFAs can lead to digester failure, it was concluded that the enzyme additives enhanced digester performance in terms of degradation of COD, reduction in sludge solids remaining after digestion and improved digester stability owing to the stable prevailing pH. The results indicate that enzyme addition at full scale could be expected to lead to greater methane yields, lower strength sludge liquors and a significant reduction in the requirements for and costs of digested sludge dewatering and disposal.
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Recombinant lamb chymosin (RLC) was prepared and tested for its potential use in cheese production. The milk clotting activity and proteolytic activity of RLC were evaluated in comparison with commercial recombinant calf chymosin (RCC), cow rennet (CR), and microbial coagulant (MC). RLC, RCC, and MC showed similar responses to pH, with a sharp increase of the coagulation time at pH 6.6 to 6.8 and decrease of curd firmness at the pH 6.5 to 6.6. In the case of CR, we observed two clear increases in the coagulation time and decreases in the curd firmness, at pH 6.4 to 6.5 and 6.6 to 6.8. Optimal clotting activity was obtained for RLC at 40 degrees C, for both CR and RCC at 45 degrees C, and for MC at 60 degrees C. The temperature instability of RLC at temperatures above 45 degrees C could constitute a benefit in making hard cheese varieties. The additon of CaCl2 to milk resulted in enhanced clotting activity of all coagulants, most prominently for CR. The proteolytic activity of RLC was significantly lower from that of CR but not significantly different from the activity of RCC. The lower proteolytic activity in the cheese made with RLC did not have negative effect on organoleptic properties. The overall quality of the cheese made with RLC was at least comparable to that of the cheese made with RCC, and both cheeses were better scored than the cheese made with CR.
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After a brief description of the properties of bioactive peptides, the proteolytic activation of the bioactive sequences from milk protein precursors is discussed. The ability of proteolytic enzymes from various sources, especially from lactic acid bacteria, to release bioactive peptides and the physiological and biotechnological significance of these peptides in dairy products are reviewed.
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Chymosin, an aspartyl proteinase, is used for curdling of milk and manufacture of cheese. We report the purification and the physicochemical properties of chymosin isolated from the abomasal tissue of buffalo calves. The enzyme preparation extracted from buffalo abomasal tissues could be purified 29-fold using anion exchange and gel filtration chromatography. The molecular weight of the purified enzyme was 35.6 kDa on SDS-PAGE. Partial N-terminal amino acid sequence of the first eight amino acid sequences of buffalo chymosin was identical to the first eight amino acid sequences of cattle chymosin. Buffalo chymosin exhibited a skewed bell-shaped stability profile as a function of temperature with maximum activity near 55 degrees C. Milk clotting activity decreased gradually as pH increased. The enzyme became completely inactive, however, above pH 7.0. The ratio of milk clotting to proteolytic activity was 3.03. When compared with cattle chymosin, there were subtle differences in the stability and relative proteolytic activity of buffalo chymosin.
Chapter
The many fermented milks familiar across the world, which have traditionally been made by spontaneous growth and action of microorganisms in the special environment afforded by milk, are today carefully controlled microbial processes for which selected cultures have been developed. The technology required for production of large quantities of the now-familiar yoghurt, cheeses, fromage frais, etc. have developed from the knowledge of the physiology and biochemistry of the microorganisms involved. The diversity of fermented milks makes a discussion of starter physiology and biochemistry complex, particularly as there has been an increase in the types of fermented milks available since publication of the first edition of this book. Products such as Laban, ‘bio-yoghurts’ and the thickened and pourable (drinking) varieties of yoghurt have appeared widely in Europe. No longer can the physiology and biochemistry be confined to a general discussion of a few species of lactococci, leuconostocs, lactobacilli and streptococci. Over the last 10 years much work has been done on the biochemistry and molecular biology of these organisms. Not only is catabolism an important consideration for a successful fermentation to produce a product of good quality in terms of flavour and stability, but anabolic pathways also have a role in providing texture-modifying polysaccharides, and in providing other compounds with a role in preservation and health-promoting properties (bacteriocins and expression of cell surface components).
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Lactoferrin is the major antimicrobial agent present in human milk, where it has a significant role in the protective effects ascribed to breast-feeding. The mechanisms of the antimicrobial effects of lactoferrin are critically discussed in relation to important bacterial and viral pathogens and the most promising areas for basic and applied research are specifically highlighted. Applications of lactoferrin in the study of the mechanisms of iron uptake by microbial pathogens, as well as in the diagnosis of gastrointestinal inflammation of infectious and autoimmune origin, are addressed. The progress in the use of lactoferrin as an immunomodulating agent are briefly discussed and the observed side effects of lactoferrin administration are singled out for consideration in clinical studies. As with the rest of the components of the innate immune system, the final outcome of the biological activity of lactoferrin is the result of combined effects with other innate and adaptive immune factors. In this regard, the availability of recombinant lactoferrin will allow clinical trials addressing the innocuity and efficacy of single or combined formulations of lactoferrin with other biologically active molecules. Diseases such as AIDS, chronic Hepatitis C, bacterial sepsis, arthritis, and bacterial infections in diabetic subjects, which lack effective treatment and have the potential to be modulated by lactoferrin, may be considered as targets for such trials. Lactoferrin research has reached a stage where a rigurous clinical evaluation of the multiple effects observed in vitro would provide a definitive answer to its actual antimicrobial and diagnostic value.
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β-Galactosidase is an important industrial enzyme in the hydrolysis of milk and whey lactose. The enzymatic hydrolysis of lactose allows to avoid health and environmental problems posed by this disaccharide. In addition, this enzyme catalyses the formation of galacto-oligosaccharides, which are prebiotic additives for the so-called "healthy foods". β-Galactosidase is one of the relatively few enzymes that have been used in large-scale processes in both free and immobilised forms. This article presents a review of recent trends in immobilisation of β-galactosidase and their application in food industry.
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Lactoferrin is an iron binding glycoprotein that contains antimicrobial peptides in its structure, which are released when hydrolysed by proteases. The antibacterial activity of bovine lactoferrin, its hydrolysates obtained with pepsin, chymosin and microbial rennet, and bovine whey fractions, has been assayed against Listeria monocytogenes serovar 4b in this study. The hydrolysates obtained with each enzyme were found to be inhibitory of bacterial growth; although the activity was lower than that exerted by the whole lactoferrin, except at low concentrations for chymosin and microbial rennet hydrolysates. The antibacterial activity of all the hydrolysates corresponded to the fraction with a molecular mass higher than 3 kDa. The peptides of hydrolysates were fractionated by using sulfonic acid derivatives and the cationic peptides analysed by matrix-assisted laser desorption ionisation-time of flight mass spectrometry. Moreover, some antibacterial activity was found in fractions obtained from bovine whey by size exclusion chromatography.
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Article
Vegetable rennet extracted from Cynara cardunculus flowers is traditionally used in the manufacture of La Serena cheese. High levels of proteolytic enzymes of the flowers are responsible for its clotting activity and strong proteolytic action. The presence of residual coagulant in cheese and whey was measured by adding known amounts of vegetable rennet as internal standard. We found no differences between the residual coagulant activity of La Serena cheese compared with other types of cheese. The coagulant content detected at the end of four cheesemakings (vat of 830 l) in cheese and whey represented 27 and 78%, respectively, of the total amount added to milk. When measurements were carried out in 16 different cheeses, vegetable rennet appeared to be highly stable during cheese ripening. Cheese composition (moisture, pH, NaCl, fat and protein) was kept relatively constant during ripening, which seems to contribute to stability of residual activity. Electrophoretic analyses of water insoluble fractions from cheeses manufactured with vegetable rennet showed that αs-casein was less susceptible to proteolysis than β-casein. The water soluble nitrogen/total nitrogen (WSN/TN) exhibited higher levels only during the first 30 days of ripening although non-protein nitrogen/total nitrogen (NPN/TN) ratio and amino acid nitrogen (NH2-N) increased with ripening time.
Article
Four lots of Canestrato Pugliese cheese were produced to evaluate the influence of the type of rennet (lamb or bovine) and of the mould size (3 and 7 kg) on the proteolysis and chemical composition of this hard cheese made from sheep milk. The curd obtained with lamb rennet showed higher retention of milk fat than the curd made with bovine rennet. This is related to the different chymosin/pepsin ratio, which influenced the state of aggregation of the casein micelles. Hydrolysis of the protein fraction during ripening was affected by the type of rennet used, due to the different retention in the curd, and by the different residual activity influenced by the physicochemical conditions of the cheeses. The highest level of proteolysis was found in the 3 kg cheese made with lamb rennet. Moreover, formation of water soluble nitrogen was closely related to the area/weight ratio of the cheeses.
Article
A chromatographic study, based on reversed-phase, size exclusion and hydrophobic interaction chromatography, was performed to evaluate the influence of weight of the cheese and type of rennet used on the evolution of soluble nitrogen in "Canestrato Pugliese", a Protected Designation of Origin (PDO) hard cheese produced in Southern Italy fromsheep milk. The weight of the cheese had only a quantitative influence, with higher amounts of soluble nitrogen in the smaller cheeses. The influence of the rennet was both quantitative and qualitative, probably due to the different chymosin/pepsin ratios. More soluble nitrogen was found in cheeses obtained with lamb rennet and significant differences were observed among the chromatographic profiles. Size exclusion chromatography revealed a significant accumulation of medium - molecular-weight peptides during ripening in the lamb rennet cheeses, whereas a faster formation of low-molecular-weight peptides was found in those produced with bovine rennet. The most distinctive peptide fraction (about 4 KD), recovered and analysed by hydrophobic interaction chromatography, revealed the presence of further qualitative differences, which were confirmed by reversed phase chromatography and free amino acid analysis. Such findings indicate a different evolution of secondary proteolysis among the cheeses. Further study is needed to better understand the mechanism of pepsin retention in the curd and its residual activity during ripening.
Article
Digestive lipases from Chinook salmon and New Zealand hoki were evaluated as flavour modifying agents in dairy products. Cream was incubated either with fish lipase or commercially available lipases used in dairy flavour development – calf pregastric esterase (Renco™ PGE) and microbial lipase (Palatase® 20,000 L). The fish enzymes were more similar to calf PGE in terms of the total amount and types of fatty acids released over the course of the reaction. Like the pregastric esterase, the fish enzymes released mainly short chain fatty acids. The highest specificity was towards the key dairy product flavour and odour compounds, butanoic and hexanoic acids. The odour intensity of hexanoic acid produced by the salmon lipase, as measured by SPME–GC–MS, was similar to that produced by both Palatase® and PGE. Free fatty acid composition, together with sensory characteristics of lipase-treated creams, demonstrated the potential for flavour enhancement in dairy products using fish lipases.
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Article
Milk clotting for the production of novel dairy products, alternative or complementary to cheese and yogurt type products can be achieved using plant sulfhydryl proteases. The objective was to apply the protease actinidin, from Actinidia chinensis, as the milk clotting agent, and High pressure (HP) technology to control excessive proteolysis. The effect of the dairy substrate and the process parameters on the coagulation rate and the texture and sensory properties of the end product, were studied. Selected values of design parameters were 25% total solids, 6.49 adjusted pH, 0.35 U activity of the clotting agent actinidin, 40ºC process temperature and 2h time. The selected pressure-temperature conditions, 600MPa at 40ºC, were applied to stop the potentially detrimental further proteolytic action of the enzyme. Results indicated that use of actinidin for milk clotting and HP to stop the enzyme activity in the final product, leads to a “fresh cheese” type dairy product.Industrial relevance: Alternative clotting methods for novel dairy products, complementary to cheese and yogurt type products, are of interest to the industry. Plant proteases can be a viable approach, provided that excessive proteolysis after structure formation is regulated. High hydrostatic pressure can be used for controlling proteolytic activity in the final products without affecting their texture and sensory characteristics.
Article
In order to optimize the process of cheesemaking of Serra cheese, the most famous traditional Portuguese cheese manufactured with raw ewes' milk at farm level, several cheeses were produced according to a second-order composite factorial design using temperature of coagulation, amount of plant rennet added and amount of salt added to fresh cheese as the manipulated technological variables. Sensory, microbiological, physicochemical and biochemical analyses were carried out for all experimental cheeses and for two cheeses manufactured by traditional practices. The amount of salt added to fresh cheese affected significantly the sensory, microbiological and biochemical characteristics of 60 day-old Serra cheese, whereas the amount of plant rennet added and the coagulation temperature affected significantly only the sensory and microbiological attributes. None of the three technological parameters at the levels tested played a statistical role at the 5% significance level in the chemical composition of the cheese. It was found that 0.3 g of plant rennet added per litre of milk, a coagulation temperature of 28 °C, and 0.05 g of salt added per cm2 of fresh cheese surface provide the best quality of cheese in terms of highest sensory scores and lowest numbers of viable enterococci and coliforms.
Article
The effectiveness of lysozyme and ethylenediaminetetraacetic disodium salt (Na2-EDTA) against the spoilage microorganisms of mozzarella cheese was studied. Mozzarella cheeses were packaged in a conditioning solution (diluted brine), which contained lysozyme (0.25mgmL−1) and different amounts of Na2-EDTA (10, 20 and 50mmolL−1), and stored at 4°C for 8 days. The population of spoilage microorganisms (total coliforms and Pseudomonadaceae), along with the functional microbiota of mozzarella cheese (lactic acid bacteria) was enumerated. Lysozyme and Na2-EDTA significantly inhibited the growth of coliforms and Pseudomonadaceae during the first 7 days of storage, whereas the functional microbiota (or lactic acid bacteria) were not affected. The results of this study showed that it is possible to extend the shelf life of mozzarella cheese through the use of lysozyme and Na2-EDTA in the conditioning brine.
Article
Roncal cheeses manufactured using lamb or calf rennet were compared for their chemical, proteolytic, and sensory characteristics. Trends in the overall physicochemical parameter values (pH, dry matter, fat, protein) were similar in both cheeses. Proteolysis of the nitrogen fractions was observed to take place faster in the cheeses made using lamb rennet (soluble nitrogen and non-protein nitrogen values around 20% higher than in the cheeses made using calf rennet up to 120d of ripening). The values for the amino acid nitrogen did not exhibit any differences according to the type of rennet used. Urea-PAGE electrophoretic analysis of the caseins from the cheeses manufactured using both types of rennet showed that the β-caseins were less susceptible to proteolysis than the α-caseins. The cheeses made using lamb rennet had lower levels of residual β- and αs-casein at the end of ripening, again an indicative of more active casein hydrolysis when lamb rennet was used. The differences in proteolysis did not yield appreciable sensory differences.
Article
Coagulating enzymes are an absolute necessity for the production of ripened cheese varieties. The objective of this review is to summarise and interpret the latest findings for the most important types of enzymes, which are animal rennet, genetically engineered chymosin, coagulants of microbial origin, and plant-derived clotting enzymes. Special emphasis has been placed on aspects of enzyme chemistry and technology, selected methods for the analysis of coagulants, and the impact of the enzymes on proteolysis, cheese yield and cheese quality.
Article
This study was conducted in order to evaluate the effects of transglutaminase (TGase) addition on some properties of set-type yoghurts such as titratable acidity, lactic acid, tyrosine, viscosity, gel firmness, syneresis, aroma compounds, sensory analysis, and micro-structural properties. The enzyme was added to yoghurt-milk at different production steps (after homogenization, after pasteurization and together with starter culture addition) and two different incubation times (10 min and 1 h) were used. Five TGase treated yoghurt samples and control sample were analyzed on 1st, 10th, and 20th days of storage. TGase addition did not cause significant changes on chemical properties of yoghurts. However, enzyme addition after pasteurization increased the gel strength and decreased the syneresis. Results of electron microscope showed that enzyme addition led proteins to be distributed more evenly in gel network due to the formation of cross-links between proteins.Graphical abstractThe use of transglutaminase (TGase) enzyme during set-type yoghurt production was studied by comparing of some properties of end products. Gas chromatography (GC) was used for some chemical analysis and scanning electron microscopy (SEM) was used to determine structural changes.
Article
Modification of proteins by enzymes such as transglutaminase (TG) has recently become of great interest to food scientists. TG (EC 2.3.2.13) catalyses the post-translational modification of proteins by transamidation of available glutamine residues by the formation of covalent cross-links between glutamine and lysine residues in proteins. It is suggested that TG is a useful tool for the production of dairy products. In this review, properties of TG and its possible use in the manufacture of dairy products are discussed. Aspects covered include reactions catalysed by the enzyme and properties of modified proteins of selected milk proteins and products.
Article
The growth of different species of the genera Lactobacillus, Streptococcus, Pediococcus and Leuconostoc was followed in media in which all available iron and copper was chelated by 2,2-dipyridyl. None of the species tested was inhibited in its growth. Iron and copper complexation, by means of chemical scavengers, could be a useful method to direct non-axenic lactic fermentations.
Article
The aim of this work was to check the efficacy of bovine lactoferrin (BLF) and its pepsin-digested hydrolysate (LFH) to control spoilage bacteria contaminating the governing liquid of high moisture (HM) Mozzarella cheese during cold storage. These natural substances resulted effective when tested in vitro against five potential spoilage bacteria contaminating cold-stored HM Mozzarella cheese. Among six LFH fractions, only the fraction containing lactoferricins, mainly represented by LfcinB₁₇₋₄₂, resulted effective against Escherichia coli K12 at the same extent of the whole pepsin-digested hydrolysate. LFH tested throughout seven days for its antimicrobial activity against the main bacterial groups growing in cold-stored commercial HM Mozzarella cheese samples delayed significantly the growth of pseudomonads and coliforms in comparison with the un-treated samples. This is the first report providing a direct evidence of the ability of LFH to inhibit the growth of cheese spoilage bacteria.
Article
The enzymes involved in cheese ripening and the factors affecting their activity in cheese is reviewed. The review begins by outlining the various enzymes that contribute to cheese ripening including those originating from milk, coagulant, starter and non-starter Lactic Acid Bacteria, and those added as exogenous preparations to accelerate flavour development. Factors influencing the retention, release and residual activity of these enzymes in cheese are discussed along with an outline of innovative methods to enhance the retention and release of exogenous enzymes.
Article
The effect of thermal treatment of whey proteins (50 g/100 g) at 75, 80, 85, 90 and 95 °C before enzymatic treatment with microbial transglutaminase was evaluated by rheological measurements. Thermal denaturation of whey proteins was determined by differential scanning calorimetry (DSC); and the gel point and turbidity were also evaluated after addition of transglutaminase at different pH values in protein solutions. A significant (p < 0.05) interaction was observed between transglutaminase and thermal treatments. At temperatures higher than 85 °C the apparent viscosity measurements of whey protein solutions with transglutaminase were significantly higher than those of the control samples. DSC analysis showed that thermal denaturation occurred at temperatures close to 82 °C, and the enzymatic reaction was enhanced at higher temperatures. The gel point of whey proteins decreased with transglutaminase addition. This decrease became greater as a function of reaction time due to the formation of high weight protein polymers catalyzed by transglutaminase, which was also observed in the turbidity analysis.
Article
The effects of rennet type (animal or vegetable) on the physicochemical characteristics (moisture, fat, protein, lactose, lactic acid, NaCl, ash, water activity, pH and nitrogen fractions: SN, NPN, AAN and NH3-N) of “Los Pedroches” cheese manufactured with pure ewes' milk were investigated. Changes were observed during 100 days of ripening. Analysis of the covariance was carried out, using these chemical composition data, expressed as a function of the dry matter. The rennet type influenced moisture, protein, and water activity values (higher in the cheeses manufactured with animal rennet), fat and SN (higher in cheeses manufactured with vegetable rennet). There was a significant rising trend in the levels of lactic acid, ash, NaCl, and nitrogen fractions during ripening, while a significant decrease was observed in the moisture, lactose, pH and aw values.
Article
Whey permeate (containing 14, 20 or 23% lactose) was used in the continuous production of oligosaccharides (OS) by hydrolysis from Maxilact 2000 L (Kluyveromyces lactis β-d-galacosidase). Two types of membrane reactors were used: a laboratory scale, Amicon stirred cell, using a flat membrane (41.8 cm2) and a pilot plant-scale membrane reactor using a Romicon hollow fiber cartridge (0.5 m2). Batch experiments were run to determine the optimal reaction time (when the largest amount of OS are produced). This optimal reaction time was then used in the experiments in continuous mode as the mean residence time of the whey permeate in the reactor, which could be controlled by adjusting the rate of permeate and or the volume of the substrate in the system.
Article
By varying cross-linking intensity, the effect of microbial transglutaminase on acid gels made from casein solution and raw milk was studied. To avoid any impact of heating, N-ethylmaleimide was used for enzyme inactivation after appropriately checking its efficiency. Up to a specific degree of oligomerisation gel stiffness and firmness increased and tan δ, time at gelation onset and syneresis decreased. Above approximately 70% and 25% of cross-linked protein in casein solution and raw milk, respectively, these parameters showed an opposite behaviour, and weak gels with high syneresis were obtained. Substrate differences, such as preferred cross-linking of adjoining κ-caseins on the surface of the micelle enhanced the effect of steric hindrance in raw milk and impaired proper rearrangements upon acidification at a much lower level of oligomerised protein. It is mainly dimeric and trimeric casein that successfully contributed to the enhanced properties of milk protein gels.
Article
In this paper, a process for the stable production of low allergenicity hydrolysates is presented. Whey protein was hydrolysed at 50 °C and pH 8.5 using a bacterial protease in a continuous stirred tank membrane reactor including a polyethersulfone plate and frame ultrafiltration module with a molecular weight cut-off of 3 kDa. The reactor was maintained in operation for 16 h. Conversion reached a steady value around 80% after 10 h of operation, while a steady permeate flow was achieved after 13 h. A slight, first order enzyme thermal inactivation was detected. A hydrolysate with an average peptide chain length around 4 amino acids was obtained. The antigenic whey protein in the product was reduced 99.97%, which suggests that it can be incorporated as nitrogen source in infant formula and enteral nutrition.
Article
The flavor of whey protein can carry over into ingredient applications and negatively influence consumer acceptance. Understanding sources of flavors in whey protein is crucial to minimize flavor. The objective of this study was to evaluate the effect of annatto color and starter culture on the flavor and functionality of whey protein concentrate (WPC). Cheddar cheese whey with and without annatto (15 mL of annatto/454 kg of milk, annatto with 3% wt/vol norbixin content) was manufactured using a mesophilic lactic starter culture or by addition of lactic acid and rennet (rennet set). Pasteurized fat-separated whey was then ultrafiltered and spray dried into WPC. The experiment was replicated 4 times. Flavor of liquid wheys and WPC were evaluated by sensory and instrumental volatile analyses. In addition to flavor evaluations on WPC, color analysis (Hunter Lab and norbixin extraction) and functionality tests (solubility and heat stability) also were performed. Both main effects (annatto, starter) and interactions were investigated. No differences in sensory properties or functionality were observed among WPC. Lipid oxidation compounds were higher in WPC manufactured from whey with starter culture compared with WPC from rennet-set whey. The WPC with annatto had higher concentrations of p-xylene, diacetyl, pentanal, and decanal compared with WPC without annatto. Interactions were observed between starter and annatto for hexanal, suggesting that annatto may have an antioxidant effect when present in whey made with starter culture. Results suggest that annatto has a no effect on whey protein flavor, but that the starter culture has a large influence on the oxidative stability of whey.
Article
The potential suitability of 10 commercial protease and lipase products for cleaning-in-place (CIP) application in the dairy industry was investigated on a laboratory scale. Assessment was based primarily on the ability of the enzymes to remove an experimentally generated milk fouling deposit from stainless steel (SS) panels. Three protease products were identified as being most suitable for this application on the basis of their cleaning performance at 40 °C, which was comparable to that of the commonly used cleaning agent, 1% NaOH at 60 °C. This was judged by quantification of residual organic matter and protein on the SS surface after cleaning and analysis by laser scanning confocal microscopy (LSCM). Enzyme activity was removed/inactivated under conditions simulating those normally undertaken after cleaning (rinsing with water, acid circulation, sanitation). Preliminary process-scale studies strongly suggest that enzyme-based CIP achieves satisfactory cleaning at an industrial scale. Cost analysis indicates that replacing caustic-based cleaning procedures with biodegradable enzymes operating at lower temperatures would be economically viable. Additional potential benefits include decreased energy and water consumption, improved safety, reduced waste generation, greater compatibility with wastewater treatment processes and a reduction in the environmental impact of the cleaning process.
Article
The negative effects on cheese quality of milk contaminated by spores of Clostridium butyricum and Cl.tyrobutyricum is prevented by the use of egg white lysozyme as additive. Since the presence of lysozyme in cheese could be possibly risky in allergic subjects, we aimed at investigating its absorption as well as serum IgE antibody titers after ingestion of Grana Padano cheese, an Italian DPO, long-ripened hard cheese, in white egg allergic subjects. Cheese lysozyme was measured by HPLC. Ten healthy volunteers and 20 patients with hen egg hypersensitivity, RAST positive (binding > or = 3%) to lysozyme and/or ovomucoid and ovalbumin received 15, 30 and 60 g of cheese at distance of at least 2 weeks each. Serum lysozyme was measured by ELISA and specific IgE binding to lysozyme by the radioallergosorbent test (RAST). The concentration of lysozyme in cheese was 155 +/- 5 mg/kg. The area-under-the-curve of serum lysozyme after 15 g of cheese was 244.5 +/- 14.0 in controls and 330.2 +/- 9.9 in patients (p < 0.01). Similar results were obtained with 30 and 60 g of cheese. Only 3 patients (15%) showed positive IgE antibody responses to cheese (overall RAST mean 4.45 +/- 1.25 % vs. 4.24 +/- 1.02 % baseline, p = ns). The amount of lysozyme absorbed with cheese was globally very low, although it was significantly lower in healthy controls than in allergic patients, where it induced an increase of IgE RAST score in 15% of subjects, without any clinical reaction. Therefore, the use of lysozyme as additive in Grana Padano cheese, does not appear to be harmful in egg allergic subjects.
Article
The iron requirement of four strains of lactobacilli (L. acidophilus, L. delbrueckii subsp. bulgaricus, L. plantarum, and L. pentosus) was studied in a synthetic medium under aerobic or anaerobic conditions. Effects of iron salt and iron-chelated compounds were tested on bacterial growth in manganese-free or -supplemented media. No significant growth stimulation was observed in any condition. These results support the absolute manganese requirement for optimum growth of lactobacilli and the needless incorporation of iron in growth media.
Article
The aim of this research is to quantify the effect of temperature and enzyme origin on the enzymatic synthesis of oligosaccharides. Quantification of these effects is important because temperature and enzyme origin are important process parameters. A kinetic model was used to describe the concentrations in time. The kinetic parameters were determined by using data obtained in batch experiments at various temperatures (20, 30, 40, and 50 degrees C) and by using beta-galactosidases from Bacillus circulans, Aspergillus oryzae, Kluyveromyces lactis, and Kluyveromyces fragilis. The effect of temperature on the kinetic parameters could be described with the Arrhenius equation, except for the inhibition parameter. Slightly higher oligosaccharide yields were found at higher temperatures. However, the influence of the initial lactose concentration was much larger. The higher yield at higher temperatures is an additional advantage when operating at high initial lactose concentrations and consequently elevated temperatures. Clear differences between the beta-galactosidases were found concerning amount, size, and type of oligosaccharides produced. The beta-galactosidase from B. circulans produced the most abundant amount, the most different, and largest-sized oligosaccharides. The beta-galactosidases from Kluyveromyces spp. produced mainly trisaccharides. The kinetic parameters for the different enzymes were determined and differences were discussed.
Article
There are a number of methods that can be used for the preparation of enzyme-containing lipid vesicles (liposomes) which are lipid dispersions that contain water-soluble enzymes in the trapped aqueous space. This has been shown by many investigations carried out with a variety of enzymes. A review of these studies is given and some of the main results are summarized. With respect to the vesicle-forming amphiphiles used, most preparations are based on phosphatidylcholine, either the natural mixtures obtained from soybean or egg yolk, or chemically defined compounds, such as DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) or POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine). Charged enzyme-containing lipid vesicles are often prepared by adding a certain amount of a negatively charged amphiphile (typically dicetylphosphate) or a positively charged lipid (usually stearylamine). The presence of charges in the vesicle membrane may lead to an adsorption of the enzyme onto the interior or exterior site of the vesicle bilayers. If (i) the high enzyme encapsulation efficiencies; (ii) avoidance of the use of organic solvents during the entrapment procedure; (iii) relatively monodisperse spherical vesicles of about 100 nm diameter; and (iv) a high degree of unilamellarity are required, then the use of the so-called 'dehydration-rehydration method', followed by the 'extrusion technique' has shown to be superior over other procedures. In addition to many investigations in the field of cheese production--there are several studies on the (potential) medical and biomedical applications of enzyme-containing lipid vesicles (e.g. in the enzyme-replacement therapy or for immunoassays)--including a few in vivo studies. In many cases, the enzyme molecules are expected to be released from the vesicles at the target site, and the vesicles in these cases serve as the carrier system. For (potential) medical applications as enzyme carriers in the blood circulation, the preparation of sterically stabilized lipid vesicles has proven to be advantageous. Regarding the use of enzyme-containing vesicles as submicrometer-sized nanoreactors, substrates are added to the bulk phase. Upon permeation across the vesicle bilayer(s), the trapped enzymes inside the vesicles catalyze the conversion of the substrate molecules into products. Using physical (e.g. microwave irradiation) or chemical methods (e.g. addition of micelle-forming amphiphiles at sublytic concentration), the bilayer permeability can be controlled to a certain extent. A detailed molecular understanding of these (usually) submicrometer-sized bioreactor systems is still not there. There are only a few approaches towards a deeper understanding and modeling of the catalytic activity of the entrapped enzyme molecules upon externally added substrates. Using micrometer-sized vesicles (so-called 'giant vesicles') as simple models for the lipidic matrix of biological cells, enzyme molecules can be microinjected inside individual target vesicles, and the corresponding enzymatic reaction can be monitored by fluorescence microscopy using appropriate fluorogenic substrate molecules.
Article
The purpose of this review is to describe the antibacterial properties and mode of action of lysozyme against gram-positive and gram-negative bacteria, and to provide insight in the underlying causes of bacterial resistance or sensitivity to lysozyme. Such insight improves our understanding of the role of this ubiquitous enzyme in antibacterial defense strategies in nature and provides a basis for the development and improvement of applications of this enzyme as an antibacterial agent. The bactericidal properties of lysozyme are primarily ascribed to its N-acetylmuramoylhydrolase enzymic activity, resulting in peptidoglycan hydrolysis and cell lysis. However, an increasing body of evidence supports the existence of a nonenzymic and/or nonlytic mode of action. Because gram-negative bacteria, including some major foodborne pathogens, are normally insensitive to lysozyme by virtue of their outer membrane that acts as a physical barrier preventing access of the enzyme, several strategies have been developed to extend the working spectrum of lysozyme to gram-negative bacteria. These include denaturation of lysozyme, modification of lysozyme by covalent attachment of polysaccharides, fatty acids and other compounds, attachment of C-terminal hydrophobic peptides to lysozyme by genetic modification, and the use of outer membrane permeabilizing agents such as EDTA or polycations or permeabilizing treatments such as high hydrostatic pressure treatment.
Article
A microtiter plate assay was employed to systematically assess the interaction between ethylenediaminetetraacetic acid (EDTA) or lactoferrin and nisin, lysozyme, or monolaurin against strains of Listeria monocytogenes, Escherichia coli, Salmonella enteritidis, and Pseudomonas fluorescens. Low levels of EDTA acted synergistically with nisin and lysozyme against L. monocytogenes but EDTA and monolaurin interacted additively against this microorganism. EDTA synergistically enhanced the activity of nisin, monolaurin, and lysozyme in tryptic soy broth (TSB) against two enterohemorrhagic E. coli strains. In addition, various combinations of nisin, lysozyme, and monolaurin with EDTA were bactericidal to some gram-negative bacteria whereas none of the antimicrobials alone were bactericidal. Lactoferrin alone (2000 microg ml(-1)) did not inhibit any of the bacterial strains, but did enhance nisin activity against both L. monocytogenes strains. Lactoferrin in combination with monolaurin inhibited growth of E. coli O157:H7 but not E. coli O104:H21. While lactoferrin combined with nisin or monolaurin did not completely inhibit growth of the gram-negative bacteria, there was some growth inhibition. All combinations of EDTA or lactoferrin with antimicrobials were less effective in 2% fat UHT milk than in TSB. S. enteritidis and P. fluorescens strains were consistently more resistant to antimicrobial combinations. Resistance may be due to differences in the outer membrane and/or LPS structure.
Article
Microbial biofilm has become difficult to control by antibiotic and biocide regimes that are effective against suspended bacteria. Their colonization of surfaces can be a problem and is generally controlled through cleaning and disinfection. This study was undertaken to examine the efficacy of the disinfectants including Bio-Ow, Econase CE, Gamanase GC 140, IndiAge 44L, Mannanase AMB, Multifect P-3000, Neutrase, Pandion, Paradigm, Pectinex Ultra SP-L, Promozyme, Resinase A2X, Spezyme AA300, Spezyme GA300 and Vinozym EC, and the proteinase against bacterial biofilms. The effectiveness of 20 commercial disinfectants against Pseudomonas aeruginosa (P. aeruginosa) biofilms using a fluorometric technique was examined. Additionally the disinfectants were also tested against Lactobacillus bulgaricus (L. bulgaricus), Lactobacillus lactis (L. lactis) and Streptococcus thermophilus (S. thermophilus) isolates using microtitration tray based turbidimetric techniques. Escherichia coli (E. coli) was used as the test bacteria in the fluorometric control method. Among the first group of the enzymatic cleaning agents tested, four disinfectants (Pandion, Resinase A2X, Spezyme GA300 and Paradigm) were the most potent against bacterial biofilms after 30 min incubation time (residual bacterial count less than 10(3) CFU (colony forming units)/ml). However, only Resinase A2X and Paradigm showed a good effect on bacterial biofilms after 15 min incubation time. Proteinase disinfectants (alkalase, chymotrypsin, cryotin and krilltrypsin) from the second group of the disinfectants showed a good effect against P. aeruginosa biofilm when tested in the absence of milk. The performance of the disinfectants was reduced in the presence of milk. The minimum inhibitory concentration (MIC) of the cleaning agents was determined as the lowest concentration inhibiting bacterial growth. The MIC was tested on Lactobacillus bulgaricus (L. bulgaricus), Lactobacillus lactis (L. lactis) and Streptococcus thermophilus (S. thermophilus) isolates. The minimum inhibitory concentrations (MIC) for Paradigm against S. thermophilus and L. Lactis were lower than L. Bulgaricus. Whereas, the MIC of Pandion against L. bulgaricus was lower than MIC against L. lactis. Resinase A2X had no inhibitory effect on bacterial growth when the concentration was less than or equal to 2.4 mg/ml and Spezyme GA 300 concentration less than or equal to 7.3 mg/ml. Minimum inhibitory concentration of Pandion against L. bulgaricus was 2.7 microg/ml and against L. lactis 5.3 microg/ml. Growth of S. thermophilus was inhibited in all concentration of Pandion tested. The choice of disinfectant or cleaning agent along with the optimum concentration and the time of action is very important when destroying microbes. It is also important that the resistances of microbes to different disinfectants and cleaning agents be taken into account when planning the cleaning process
Article
The research field for applications for lactose hydrolysis has been investigated for some decades. Lactose intolerance, improvement for technical processing of solutions containing lactose and utilisation of lactose in whey are main topics in development of biotechnological processes. In this article, the establishment of a hollow fiber membrane reactor process for enzymatic lactose hydrolysis is reported. Mesophilic beta-galactosidases were circulated abluminally during luminal flow of skim milk. The main problem, microorganisms growth in the enzyme solution, was minimised by sterile filtration and UV irradiation. In order to characterise the process parameters, such as skim milk concentration, enzyme activity and flow rates were varied. In comparison to a batch process, enzyme activity could be used longer and enzyme rest into the product should not occur. Furthermore, the three-dimensional separation of the substrate from the enzyme solution minimise blocking and washing out effects, which restrict processes with immobilised enzymes. A conversion rate of 78.11% was achieved at a skim milk flow rate of 9.9l h(-1), enzyme activity of 120 Uml(-1) and a temperature of 23+/-2 degrees C in a hollow fiber reactor with a membrane area of 4.9 m2.
Article
Enzymatic milk coagulation for cheese manufacturing involves the cleavage of the scissile bond in kappa-casein by an aspartic acid protease. Bovine chymosin is the preferred enzyme, combining a strong clotting activity with a low general proteolytic activity. In the present study, we report expression and enzymatic properties of recombinant camel chymosin expressed in Aspergillus niger. Camel chymosin was shown to have different characteristics than bovine chymosin. Camel chymosin exhibits a 70% higher clotting activity for bovine milk and has only 20% of the unspecific protease activity for bovine chymosin. This results in a sevenfold higher ratio of clotting to general proteolytic activity. The enzyme is more thermostable than bovine chymosin. Kinetic analysis showed that half-saturation is achieved with less than 50% of the substrate required for bovine chymosin and turnover rates are lower. While raw camel milk cannot be clotted with bovine chymosin, a high clotting activity was found with camel chymosin.
Article
Wastewater from dairies and slaughterhouses contains high levels of fats and proteins that present low biodegradability. A large number of pretreatment systems are employed to remove oil and grease (O&G) to prevent a host of problems that may otherwise arise in the biological process, and reduce the efficiency of the treatment station. Problems caused by excessive O&G include a reduction in the cell-aqueous phase transfer rates, a sedimentation hindrance due to the development of filamentous microorganisms, development and flotation of sludge with poor activity, clogging and the emergence of unpleasant odors. Therefore the application of a pretreatment to hydrolyze and dissolve lipids may improve the biological degradation of fatty wastewaters, accelerating the process and improving time efficiency. However thus far, only a few studies describing the degradation of fats and oils by alkaline/acid/enzymatic hydrolysis have been reported; the treatment of effluents from several origins is a new and promising application for lipases. Among the strains that produce the hydrolytic enzymes studied, the fungus Penicillium restrictum is a particularly promising one. When cultivated in low-cost solid medium composed of agro-industrial waste, P. restrictum produces a pool of hydrolases capable of degrading the most complex organic compounds. This degradation enables a considerable increase in organic matter removal efficiency to be realized, which results in the attainment of a high-quality effluent in the subsequent biological treatment stage. Consequently, there is presently a wide variety of ongoing scientific investigation in the field of developing enzymatic hydrolysis processes to precede traditional biological treatment.