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Microstructure in mature Cheddar cheese: the effects of elevated ripening temperature, milk protein concentration, calcium chloride addition, draining pH and rennet concentration

© 2015 Dr. Kevany SoodamCheddar cheese manufacture accounts for nearly half of Australian cheese produced annually; the process must therefore be optimised for maximum economic benefits. Changes to the manufacturing or ripening conditions can greatly influence the development of the texture and flavour of the cheese during ripening. This thesis aims to use confocal laser scanning microscopy (CLSM) and cryo-scanning electron microscopy (cryo-SEM), coupled with textural and biochemical analyses, to investigate the impact of changing selected process variables on the quality of the cheese during ripening. The impact of ripening conditions was first considered, particularly the use of different ripening temperatures as it is easy for industries to implement this method. Ripening at elevated temperatures can accelerate cheese ripening and decrease energy and storage requirements, especially as Cheddar cheese ripening is a lengthy process that can last from weeks to years. The effect of elevated temperature on the biochemistry and microstructure within the cheese, however, is not well understood. Cheddar cheese was matured at four different ripening temperatures: 8°C, 15°C, 20°C or a combination of 8°C and 15°C. These temperatures were selected based on the literature as 8°C is a standard ripening temperature and 15°C and 20°C are often used to accelerate ripening. Ripening above 20°C may result in too soft texture. The ripening temperature was shown to alter the protein network of the cheese after ripening for only a few weeks. The number of vertices in the protein network, determined by three-dimensional CLSM image analysis, was lower at higher ripening temperatures early in the ripening process, consistent with the thicker and less numerous protein strands observed for these cheeses. Unexpectedly, the microstructure of the fat was not affected by elevated ripening temperatures. After 194 days of ripening, however, the cheeses ripened at 15°C or 20°C had significantly higher bacterial population than the cheeses ripened at 8°C or 15°C followed by 8°C. Ripening at higher temperatures also accelerated the ripening and thus long periods of ripening at these temperatures may have an adverse effect on the texture. Ripening the cheese at an elevated temperature for only 33 days, however, may be beneficial as this timeframe provides the acceleration of ripening desired, without affecting the texture too negatively. This study may prove useful to the dairy community, as it shows the ripening treatments that might be employed to minimise ripening time whilst still providing cheese with a desired texture. The next variables considered related to the manufacturing process. Ultrafiltration retentate may be used to standardise cheese milk and the effect of varying the milk protein concentration using low concentration factor ultrafiltration retentate (~3.7%, 4%, 4.8%, 5.8% w/w protein) on Cheddar cheese during ripening was investigated. Quantitative analysis of the rendered protein network showed a decrease in the number of vertices with ripening time, possibly indicating the solubilisation of protein. This decrease was significantly correlated with both proteolysis and texture. After ripening for 26 weeks, the cheeses made from the 4.8% w/w and 5.8% w/w milk protein were significantly harder than the cheese with no ultrafiltration retentate (UF) addition, which might appeal for particular end uses and selected consumers; the difference was no longer significant, however, after a further period of ripening. The cryo-SEM images at week 26 could also indicate a possible link between the milk protein concentration, the stretchability of the melted cheese and the microstructure of the cheese. Generally, it was observed that milk protein concentration has little effect on the cheese microstructure and texture especially when an adequate ripening period is provided; this suggests that a higher throughput of cheese may be achieved with the addition of protein if the maturation period is adequate. Calcium chloride is commonly added to cheese milk to improve the formation of the coagulum and to increase cheese yield but high concentrations of calcium ions can have adverse effects. A lower draining pH, however, could potentially be used to control the final calcium content in the cheese. The impact of adding calcium chloride (0, 100, 300 mg/kg milk) in combination with alterations to the draining pH (pH 6.0, 6.2) was investigated in this thesis. The cheeses generally ripened normally, with few significant differences resulting from the manipulation of calcium levels and the draining pH. The impact of the draining pH on the number of vertices during ripening was reduced with added calcium chloride and cheeses were harder when drained at a lower pH, possibly due to lower moisture content. The results reported in this thesis show that calcium chloride addition can be used, together with a lower draining pH, to alter the cheese manufacturing process without significantly impacting on the quality of the mature cheese. One point of interest, however, was that the cheeses drained at a lower draining pH did not show higher proteolysis, possibly due to the usage of rennet of microbial origin instead of calf rennet. Cheddar cheese made from microbial rennet, produced from Rhizomucor miehei (Hannilase), was next compared to cheese made from camel chymosin produced by fermentation. The impact of varying the concentration of microbial rennet (0.026, 0.052 and 0.150 IMCU/g of milk) was also investigated in this thesis. A high rennet concentration was also investigated, despite the higher operating cost, in order to determine whether there is any benefit, such as faster texture development. The gel made with a high rennet concentration was more porous qualitatively, possibly due to the higher speed of gel formation but the structure of the fresh cheese was not different between treatments. After 31 weeks or ripening, however, the cheese made with recombinant camel chymosin had a thicker protein network compared to the cheese made with microbial rennet, as shown by cryo-SEM, possibly due to lower proteolysis. Most of the Hannilase rennet was lost when the rennet was increased above the concentration of 0.052 IMCU/g of milk. The results show, however, that using a lower concentration of rennet may be effective as these conditions require a lower rennet dosage and the texture was not significantly affected at the end of the observed ripening period. Recombinant camel chymosin may also potentially be used as a substitute for products requiring lower proteolysis during ripening. The texture of the cheese was harder, however, than the cheese made with Hannilase rennet at the end of the ripening period. In summary, process variables affect the microstructure as well as textural and biochemical properties of the cheese to a different extent during ripening. The cheese can be ripened at an elevated temperature for a short period of time, the milk protein concentration can be increased, calcium can be added to the cheese milk, combined with a lower draining pH and the rennet concentration can potentially be decreased. The results presented in this thesis are useful for the Australian dairy industry as well as researchers from the international community. They improve our knowledge of the impact of select process parameters on the microstructure, textural and biochemical properties of cheese during ripening

Effect of elevated temperature on the microstructure of full fat Cheddar cheese during ripening

Elevated temperatures have been widely studied as a route to accelerate cheese ripening and decrease energy and storage requirements but the impact of temperature on the underlying microstructure of the cheese during prolonged periods of ripening is poorly understood. In this study, Cheddar cheese was matured at four different ripening temperatures (8 °C, 15 °C, 20 °C or a combination of 8 °C and 15 °C) and the impact on cheese microstructure assessed using confocal laser scanning microscopy, cryo scanning electron microscopy and quantitative image analysis of 3D images. An increase in ripening temperature was shown to alter the microstructure of the cheese protein network after only a few weeks of ripening. Incubation at 20 °C significantly reduced branching within the protein network, leading to thicker protein strands and larger pores after 33 days. These structural changes coincided with increased proteolysis, consistent with solubilisation of the protein network; they also led to a softer, less chewy and less cohesive cheese. While the concentration of biogenic amines tryptamine and tyramine were observed to increase with ripening temperature, the concentrations were generally low, confirming that biogenic amines do not represent a health concern under the conditions examined. This study illustrates how 3D image analysis can be used to observe and quantify the effect of process changes on cheese structure, assisting our understanding of the link between structure and function in Cheddar cheese

The addition of calcium chloride in combination with a lower draining pH to change the microstructure and improve fat retention in Cheddar cheese

Calcium chloride addition and the whey draining pH are known to impact on cheese making. The effect of 100 or 300 mg kg−1 calcium chloride (CaCl2) and the whey draining pH (6.2 or 6.0) on the microstructure of Cheddar cheese was assessed using confocal and cryo scanning electron microscopy. The gel made with 300 mg kg−1 CaCl2 was found to have a denser protein network and smaller pores than the gel with lower or no CaCl2 addition. CaCl2 addition reduced fat lost to the sweet whey. The texture of the cheeses with a lower draining pH was harder and moisture content lower. Our results show that the combination of calcium addition and lower draining pH could be used to increase network formation at the early stages of cheese making to improve fat retention while maintaining a similar level of total calcium in the final cheese