Rennet-coagulated cheeses are ripened for periods ranging from about two weeks to two or more years depending on variety. During ripening, microbiological and biochemical changes occur that result in the development of the flavour and texture characteristic of the variety. Biochemical changes in cheese during ripening may be grouped into primary (lipolysis, proteolysis and metabolism of residual lactose and of lactate and citrate) or secondary (metabolism of fatty acids and of amino acids) events. Residual lactose is metabolized rapidly to lactate during the early stages of ripening. Lactate is an important precursor for a series of reactions including racemization, oxidation or microbial metabolism. Citrate metabolism is of great importance in certain varieties. Lipolysis in cheese is catalysed by lipases from various source, particularly the milk and cheese microflora, and, in varieties where this coagulant is used, by enzymes from rennet paste. Proteolysis is the most complex biochemical event that occurs during ripening and is catalysed by enzymes from residual coagulant, the milk (particularly plasmin) and proteinases and peptidases from lactic acid bacteria and, in certain varieties, other microorganisms that are encouraged to grow in or on the cheese. Secondary reactions lead to the production of volatile flavour compounds and pathways for the production of flavour compounds from fatty acids and amino acids are also reviewed.

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Biochemistry of Cheese Ripening

Nomenclature of Proteins of Cow's Milk: Fifth Revision

Milk proteins are natural vehicles for bioactives. Many of their structural and physicochemical properties facilitate their functionality in delivery systems. These properties include binding of ions and small molecules, excellent surface and self-assembly properties; superb gelation properties; pH-responsive gel swelling behavior, useful for programmable release; interactions with other macromolecules to form complexes and conjugates with synergistic combinations of properties; various shielding capabilities, essential for protecting sensitive payload; biocompatibility and biodegradability, enabling to control the bioaccessibility of the bioactive, and promote its bioavailability. The review highlights the main achievements reported in the last 3 years: harnessing the casein micelle, a natural nanovehicle of nutrients, for delivering hydrophobic bioactives; discovering unique nanotubes based on enzymatic hydrolysis of α-la; introduction of novel encapsulation techniques based on cold-set gelation for delivering heat-sensitive bioactives including probiotics; developments and use of Maillard reaction based conjugates of milk proteins and polysaccharides for encapsulating bioactives; introduction of β-lg–pectin nanocomplexes for delivery of hydrophobic nutraceuticals in clear acid beverages; development of core-shell nanoparticles made of heat-aggregated β-lg, nanocoated by beet-pectin, for bioactive delivery; synergizing the surface properties of whey proteins with stabilization properties of polysaccharides in advanced W/O/W and O/W/O double emulsions; application of milk proteins for drug targeting, including lactoferrin or bovine serum albumin conjugated nanoparticles for effective in vivo drug delivery across the blood-brain barrier; beta casein nanoparticles for targeting gastric cancer; fatty acid-coated bovine serum albumin nanoparticles for intestinal delivery, and Maillard conjugates of casein and resistant starch for colon targeting. Major future challenges are spot-lighted.

Milk proteins as vehicles for bioactives

Proteins are widely used as emulsifiers to facilitate the formation, improve the stability and provide specific physicochemical properties to oil-in-water emulsions. There have been a number of recent advances in the understanding of the ability of various types of proteins to provide these functional properties. This article focuses on the influence of solution composition (pH, ionic strength, sugars, polyols, surfactants, biopolymers) and environmental stresses (heating, chilling, freezing, drying) on the stability of globular protein stabilized emulsions.

Protein-stabilized emulsions

The commercially available LIVE/DEAD BacLight kit is enjoying increased popularity among researchers in various fields of microbiology. Its use in combination with flow cytometry brought up new questions about how to interpret LIVE/DEAD staining results. Intermediate states, normally difficult to detect with epifluorescence microscopy, are a common phenomenon when the assay is used in flow cytometry and still lack rationale. It is shown here that the application of propidium iodide in combination with a green fluorescent total nucleic acid stain on UVA-irradiated cells of Escherichia coli, Salmonella enterica serovar Typhimurium, Shigella flexneri, and a community of freshwater bacteria resulted in a clear and distinctive flow cytometric staining pattern. In the gram-negative bacterium E. coli as well as in the two enteric pathogens, the pattern can be related to the presence of intermediate cellular states characterized by the degree of damage afflicted specifically on the bacterial outer membrane. This hypothesis is supported by the fact that EDTA-treated nonirradiated cells exhibit the same staining properties. On the contrary, this pattern was not observed in gram-positive Enterococcus faecalis, which lacks an outer membrane. Our observations add a new aspect to the LIVE/DEAD stain, which so far was believed to be dependent only on cytoplasmic membrane permeability.

Assessment and interpretation of bacterial viability by using the LIVE/DEAD BacLight Kit in combination with flow cytometry.

1. Whey and Whey Products Whey can be defined in a very general sense as "the liquid remaining after removal of casein from milk". Casein can be separated from milk in a variety of ways resulting in the production of a number of different types of whey, each of which can be identified on the basis of the method of casein removal. From an industrial viewpoint there are two principal types of whey (IDF, 1978; Nielsen, 1974); sweet and acid wheys. These are by-products of cheese or rennet casein and acid casein manufacture, respectively. Acidification may be by direct addition of mineral acid or by in situ production of acid by added starter bacteria Sweet whey has a minimum pH of 5.6 and acid whey a maximum pH of 5.1 (IDF, 1978). Whey composition varies depending on milk source, processing methods used and the cheese or casein type but typically it contains ~ 50% of the total solids of the original milk and includes lactose, proteins, minerals and vitamins. Data on whey composition include those by Roeper (1971), Cerbulis, Woychik and Wondolowski (1972), Josephson, Rizvi and Harper (1975), Glass and Hedrick (1976a, b) and Marshall (1982). Table 1 lists the average composition of some types of whey. Whey was traditionally regarded as a waste product and was generally disposed of as effluent or as an animal feed. However, it has now come to be regarded as a product whose valuable constituents can be constructively utilized by the food industry, where there has been increased recognition that the proteins, lactose and possibly even the salts in whey are valuable nutrients. Impetus for whey utilization is due to the increased use of functional proteins in processed foods and to the development of new technologies for the economic recovery of such protein from whey. Heating has direct relevance in the development of whey processing techniques and influences the functionality of whey protein products; it is a major technological treatment, either as a process per se or as part of a process. Heating induces changes in whey constituents, especially the proteins, which have a major influence on the efficiency of the process and on the quality of the product. Thus, heating has direct effects on both the structure and functionality of these proteins in food systems. An understanding of the effects of heat on proteins, i.e. denaturation and aggergation, might permit manipulation and control of these phenomena in order to permit greater product and process control and/or to develop new processes or products with predictable or tailor-made functional properties. For example, Richert, Morr and Cooney (1974) and Richert (1979) showed that controlled heating, to induce limited 43

Whey Proteins and their Thermal Denaturation - A Review

Heating β-lactoglobulin solutions at pH 8 causes an increase in viscosity, but self-supporting gels were not formed unless salts, such as sodium chloride or calcium chloride, were added. The rheological and textural properties and gel strength were markedly affected by salt concentration. Thus, gels of maximum compressive strength were obtained with sodium chloride and calcium chloride at concentrations of 200 and 10mM, respectively. Increasing the concentration of sodium chloride resulted in the formation of soft gels which released water easily. Calcium chloride strengthened β-lactoglobulin gels by forming crossbridges. However, above 10 mM it tended to enhance coagulation rather than gelation. This was confirmed by election microscopy of the gel matrix.

Gelation of β-Lactoglobulin: Effects of Sodium Chloride and Calcium Chloride on the Rheological and Structural Properties of Gels

Denaturation and aggregation processes in thermal gelation of whey proteins resolved by differential scanning calorimetry

Confocal scanning laser microscopy (CSLM) methods were developed to identify fat and protein in cheeses, milk chocolate and milk powders. Various fluorescent probes were assessed for their ability to label fat or protein in selected food products in situ. Dual labelling of fat and protein was made possible by using mixtures of probes. Selected probes and probe mixtures were then used to study (a) structure development of Mozzarella cheese during manufacture and ripening, and (b) the distribution of fat and protein in milk chocolate made with milk powders containing varying levels of free fat. Microstructural changes in the protein and fat phases of Mozzarella cheese were observed at each major step in processing. Aggregation of renneted micelles occurred during curd formation: this was followed by amalgamation of the para-casein into linear fibres during plasticization. Following storage, the protein phase of the Mozzarella became more continuous; entrapping and isolating fat globules. Chocolate made with a high free-fat spray-dried powder blend showed a homogeneous fat distribution, similar to that of chocolate made with roller-dried milk. Chocolate made with whole milk powder containing 10 g free fat/100 g fat showed a non-homogeneous fat distribution with some fat occluded within milk protein particles. These differences in fat distribution were related to Casson yield value and Casson viscosity of the chocolates.

Development and application of confocal scanning laser microscopy methods for studying the distribution of fat and protein in selected dairy products.

The viability of the human probiotic strains Lactobacillus paracasei NFBC 338 and Bifidobacterium sp. strain UCC 35612 in reconstituted skim milk was assessed by confocal scanning laser microscopy using the LIVE/DEAD BacLight viability stain. The technique was rapid (<30 min) and clearly differentiated live from heat-killed bacteria. The microscopic enumeration of various proportions of viable to heat-killed bacteria was then compared with conventional plating on nutrient agar. Direct microscopic enumeration of bacteria indicated that plate counting led to an underestimation of bacterial numbers, which was most likely related to clumping. Similarly, LIVE/DEAD BacLight staining yielded bacterial counts that were higher than cell numbers obtained by plate counting (CFU) in milk and fermented milk. These results indicate the value of the microscopic approach for rapid viability testing of such probiotic products. In contrast, the numbers obtained by direct microscopic counting for Cheddar cheese and spray-dried probiotic milk powder were lower than those obtained by plate counting. These results highlight the limitations of LIVE/DEAD BacLight staining and the need to optimize the technique for different strain-product combinations. The minimum detection limit for in situ viability staining in conjunction with confocal scanning laser microscopy enumeration was approximately 10(8) bacteria/ml (equivalent to approximately 10(7) CFU/ml), based on Bifidobacterium sp. strain UCC 35612 counts in maximum-recovery diluent.

Direct in situ viability assessment of bacteria in probiotic dairy products using viability staining in conjunction with confocal scanning laser microscopy.

Daniel M. Mulvihill

Papers

Whey Proteins and their Thermal Denaturation - A Review

Gelation of β-Lactoglobulin: Effects of Sodium Chloride and Calcium Chloride on the Rheological and Structural Properties of Gels

Denaturation and aggregation processes in thermal gelation of whey proteins resolved by differential scanning calorimetry

Development and application of confocal scanning laser microscopy methods for studying the distribution of fat and protein in selected dairy products.

Direct in situ viability assessment of bacteria in probiotic dairy products using viability staining in conjunction with confocal scanning laser microscopy.