Pasta Filata Research Articles

Effects of different freezing methods on some properties of a pasta filata cheese

The aim of the study was to select the most suitable freezing and thawing method for preserving the quality of pasta flata type Parenica cheese comparing different methods: slow, shock (circulated air) and cryogenic freezing and thawing at room temperature and in a refrigerator, respectively. To observe the effects of these methods on some cheese properties weight, pH, and dry-matter content were measured, stringiness was examined, and compression test were performed for texture analysis. Thermophysical properties were determined by DSC: unfreezable water content, onset point of melting, and latent heat. Sensory analyses were also carried out by profle analysis. Results of objective and subjective measurements showed similar trends: characteristics of cheese samples frozen in circulated air and thawed in refrigerator resembled the most to the control sample.

Effect of pH on technological parameters and physicochemical and texture characteristics of the pasta filata cheese Telita

The objective of this study was to determine the effect of stretching pH on technological parameters and physicochemical and texture characteristics of the pasta filata cheese Telita. A no-brine cheese-making method was used to control both melting and stretching temperatures. Six vats of cheese, each with a different stretching pH (5.2, 5.3, 5.4, 5.5, 5.6, and 5.7), were made in 2h. Cheese-making was replicated using 2 different lots of milk. Differences in stretching pH significantly affected all variables evaluated; stretching temperature and pH were positively correlated. Technological parameters showed an inverse relationship between pH and acidity and a direct relationship between melting and stretching temperature. The yield was highest as the pH increased and ranged from 11.4 to 12.9kg of cheese/100kg of milk. Physicochemical characteristics showed the following: moisture 48.1 to 53.5% (soft and semi-hard cheese), fat 46.3 to 54.9% (dry basis, full-fat cheese), minerals 2.8 to 3.5% (dry basis), calcium content 0.5 to 1.0% (dry basis), sodium 0.38 to 0.78% (dry basis), and whiteness index 77.2 to 84.5. Texture parameters showed that as the stretching pH increased, hardness increased, adhesiveness decreased, cohesiveness decreased, springiness increased, and chewiness increased. Samples were grouped based on principal component analysis. Group 1 contained cheeses at pH 5.2 and 5.3 and were better in terms of retention of components. Group 2 contained cheeses at pH 5.6 and 5.7. These cheeses attained the highest yields, were whitest, and presented the highest values for texture parameters except for adhesiveness and cohesiveness. The third group of cheeses at pH 5.4 and 5.5 were considered the best because they showed a good balance among all variables evaluated.

Influence of ripening conditions on Scamorza cheese quality.

Scamorza is a pasta filata cheese produced in Southern Italy and eaten after a short ripening. The ripening phase is critical in defining the main qualitative features of the Scamorza cheese. The success of this operation is conditioned not only by the process parameters, but also by the characteristics of the ripening room in which different microclimates originate. This work intended to evaluate the influence of the different positions of cheeses within the ripening room on the evolution of their qualitative characteristics during the process of drying/ripening. For this purpose, samples of Scamorza cheese, produced in the Molise Region (Italy), were divided into two batches (C and L) and subjected to ripening for seven days in a thermo thermo-regulated room. The two batches were placed in different points of the room: the batch C in the central area and the batch L in the lateral area. During the ripening, temperature, humidity and air flow were monitored and the Scamorza cheeses were analysed to assess some qualitative characteristics. In a ripening room, the created microclimates are able to influence the quality of the product, as demonstrated by data related to temperature, humidity and air flow. In fact, from the results obtained, some appreciable differences among products from batches C and L were observed for the weight loss, the water activity and the colorimetric indexes. Differences in the behaviour of mesophilic lactic acid bacteria, pH and acidity were also found. The more rapid loss of water, characterizing the batch C, resulted in an evolution of physicochemical, physical and microbiological features, which resulted different from those observed in the samples from the batch L. Therefore, the results obtained in this study point out that, within the ripening room, the formation of different micro-environments is able to strongly influence the definition of the qualitative characteristics of the products placed in it.

Physicochemical properties of Scamorza ewe milk cheese manufactured with different probiotic cultures

The present study was undertaken to produce functional Scamorza cheese from Gentile di Puglia ewe milk by incorporating probiotic strains into the cheese matrix and to evaluate the physicochemical characteristics of Scamorza ewe milk cheese. Gentile di Puglia ewe bulk milk was used for Scamorza cheese production. Cheeses were denoted S-CO for control Scamorza cheese, S-BB for Scamorza cheese made using a mix of Bifidobacterium longum and Bifidobacterium lactis, and S-LA for Scamorza cheese made using Lactobacillus acidophilus as probiotic strain. Cheeses were analyzed at 1, 7, and 15 d of ripening. Probiotic cell recovery in cheese was 7.55±0.07 log10cfu/g and 9.09±0.04 log10cfu/g in S-LA and S-BB cheese, respectively; probiotic cheeses also displayed the highest levels of lactic microflora. Reverse-phase HPLC chromatograms of the water-soluble nitrogen fraction showed a more complex profile in S-BB, with distinctive peaks in the early-eluting zone. The matured Scamorza cheese containing the mix of B. longum and B. lactis was characterized by significantly higher levels of Gln, Ser, Arg, Ile, and Leu, whereas cheese containing Lb. acidophilus was characterized by higher levels of Tyr and Met. Total FFA content was the highest in S-LA, intermediate in S-BB, and the lowest in S-CO cheese; in particular, Scamorza cheese containing Lb. acidophilus showed the highest level of vaccenic acid, oleic acid, and total conjugated linoleic acid. Probiotic bacteria survived through the technological phases of pasta filata cheese production, maintained their specific metabolic pathways, and conferred functional properties to Scamorza ewe milk cheese.

Composition and sensory profiling of probiotic Scamorza ewe milk cheese

The present study aimed to assess the effect of the addition of different usually recognized as probiotic bacterial strains on chemical composition and sensory properties of Scamorza cheese manufactured from ewe milk. To define the sensory profile of Scamorza cheese, a qualitative and quantitative reference frame specific for a pasta filata cheese was constructed. According to the presence of probiotic bacteria, cheeses were denoted S-BB for Scamorza cheese made using a mix of Bifidobacterium longum 46 and Bifidobacterium lactis BB-12, and S-LA for Scamorza cheese made using Lactobacillus acidophilus LA-5. The designation for control Scamorza cheese was S-CO. Analyses were performed at 15d of ripening. The moisture content of Scamorza ewe milk cheese ranged between 44.61 and 47.16% (wt/wt), showing higher values in S-CO and S-BB cheeses than in S-LA cheese; the fat percentage ranged between 25.43 and 28.68% (wt/wt), showing higher value in S-LA cheese. The NaCl percentage in Scamorza cheese from ewe milk was 1.75±0.04% (wt/wt). Protein and casein percentages were the highest in Scamorza cheese containing a mix of bifidobacteria; also, the percentage of the proteose-peptone fraction showed the highest value in S-BB, highlighting the major proteolysis carried out by enzymes associated with B. longum and B. lactis strains. Texture and appearance attributes were able to differentiate probiotic bacteria-added cheeses from the untreated control product. In particular, S-BB and S-LA Scamorza cheeses showed higher color uniformity compared with S-CO cheese. Furthermore, the control cheese showed higher yellowness and lower structure uniformity than S-BB. The control product was less creamy and grainy than S-BB; conversely, the inclusion of probiotics into the cheese determined lower adhesivity and friability in S-BB and S-LA than in S-CO. This study allowed the definition of the principal composition and sensory properties of Scamorza ewe milk cheese. The specific quantitative vocabulary for sensory analysis and reference frame for assessor training also established in this study should be implemented to systematically monitor the quality of this new typology of ewe milk cheese.

Survival of microencapsulated probiotic Lactobacillus paracasei LBC-1e during manufacture of Mozzarella cheese and simulated gastric digestion

An erythromycin-resistant strain of probiotic Lactobacillus paracasei ssp. paracasei LBC-1 (LBC-1e) was added to part-skim Mozzarella cheese in alginate-microencapsulated or free form at a level of 108 and 107cfu/g, respectively. Survival of LBC-1e and total lactic acid bacteria (LAB) was investigated through the pasta filata process of cheese making (in which the cheese curd was heated to 55°C and stretched in 70°C-hot brine), followed by storage at 4°C for 6wk and simulated gastric and intestinal digestion. This included incubation in 0.1 M and 0.01 M HCl, 0.9 M H3PO4, and a simulated intestinal juice consisting of pancreatin and bile salts in a pH 7.4 phosphate buffer. Some reductions were observed in both free and encapsulated LBC-1e during heating and stretching, with encapsulated LBC-1e surviving slightly better. Changes in total LAB losses during heating and stretching did not reach statistical significance. During storage, a decrease was observed in total LAB, but no statistically significant decrease was observed in LBC-1e. Survival during gastric digestion in HCl was dependent on the extent of neutralization of HCl by the cheese, with more survival in the weaker acid, in which pH increased to 4.4 after cheese addition. The alginate microcapsules did not provide any protection against the HCl. It is interesting that survival of the encapsulated LBC-1e was greater during incubation in H3PO4 than in the HCl gastric juices. Proper selection of simulated gastric digestion media is important for predicting the delivery of probiotic bacteria into the human intestinal tract. Neither free nor encapsulated LBC-1e was affected by incubation in the pancreatin-bile solution. Based on the level of probiotic bacteria in cheese needed to provide a health benefit and its survival during simulated gastric digestion, as determined in this study, it should theoretically be possible to lower the amount that needs to be ingested in cheese by up to a factor of 103 compared with other fermented dairy foods or when consumed as supplements.

Multi-detection of preservatives in cheeses by liquid chromatography–tandem mass spectrometry

The incorrect use of preservatives in cheeses may compromise food safety and damage consumers. According to the law, more than one preservative may be contemporarily used in cheeses. So a method for their contemporary detection may be useful for both manufacturers and control agencies quality control. In this research a liquid chromatography–tandem mass spectrometric with electrospray ionization method for the multi-determination of seven preservatives (benzoic acid, citric acid, hexamethylenetetramine, lysozyme, natamycin, nisin and sorbic acid) in cheese was developed. The preservatives were contemporarily extracted from cheese by a single procedure, and analyzed by RP-LC/ESI-MS/MS (Ion Trap) in positive ionization mode, with single reaction monitoring (SRM) acquisition. Three sample types (hard, pasta filata and fresh cheese) were used for method evaluation. Recoveries were mostly higher than 90%; MDLs ranged from 0.02 to 0.26mgkg−1, and MQLs were included between 0.07 and 0.88mgkg−1. Due to matrix effect, quantitation was performed by referring to a matrix matched calibration curve, for each cheese typology. This method was also applied to commercial cheese samples, with good results. It appears fast, reliable and suitable for both screening and confirmation of the presence and quantitation of the preservatives in a single, multi-detection analysis.

An innovative pre-ripening drying method to improve the quality of pasta filata cheeses

In order to optimise the pre-ripening of pasta filata cheeses, two traditional Italian cheeses, Caciocavallo and Scamorza, were dried for different times at 15°C, 50% relative humidity and airflow rate of 1000 m3/h, by using a pre-ripening plant. During ripening, microbiological, chemical-physical and sensorial analyses were applied to the products in order to evaluate the impact of the innovative pre-ripening technique used on the cheese characteristics. The used plant led to commercial time saving and to the standardisation of the process, making it unaffected by the effects of temperature and humidity variations, common in small industries. The final products showed good chemical-physical and sensorial qualities and resulted in a higher microbiological safety, preserving their traditional characteristics.

Thin agar layer- versus most probable number-PCR to enumerate viable and stressed Escherichia coli O157:H7 and application in a traditional raw milk pasta filata cheese

A mid-log phase broth culture of Escherichia (E.) coli O157:H7 381 (final concentration 104cfu/mL) was monitored by conventional liquid- and solid‐based enumeration techniques combined with PCR while it was subjected to thermal stress in gradually more complex systems (i.e., Tryptone Soya Broth, pasteurized milk and during lab-scale productions of a pasta filata fior di latte cheese obtained from raw or pasteurized milk). Our results highlighted: i) the incapability of the selective medium, ii) the effectiveness of the thin agar layer-PCR method, and iii) the effectiveness of the most probable number (MPN)-PCR method (in comparison with both plating-based methods) in recovering and selectively counting viable and stressed or injured E. coli O157:H7. Moreover, MPN-PCR was superior to both plating-based methods in terms of speed and easiness to get results. The thermal stresses herein applied (heating at 55°C for 5 and 8min) were less effective on the pasteurized milk than on the Tryptone Soya Broth and the pathogen was more protected in the raw milk-based matrices than in the pasteurized ones. Moreover, given the contamination level (104cfu/mL of milk) of the strain, the temperature/time of stretching and the hardening and brining conditions herein used, the complete inactivation of the pathogen is not achievable.

Behaviour of Salmonella Typhimurium during production and storage of artisan water buffalo Mozzarella cheese

Water buffalo mozzarella cheese (WBMC) is a fresh pasta filata cheese produced from whole chilled buffalo milk. Although pasteurization of milk and the use of defined starter cultures are recommended, traditional technology involving the use of unpasteurized milk and natural whey cultures is still employed for WBMC production in Italy. The aim of this study were to assess the behaviour of Salmonella Typhimurium during the production of artisan water buffalo mozzarella cheese and during its shelf life under different temperature conditions. Raw milk was inoculated with S. Typhimurium and the evolution of S. Typhimurium count during production and shelf life was monitored. In artisan WBMC pro-duction technology S. Typhimurium multiplied in the curd during ripening, but its growth rate expressed in log CFU/g/h was lower than the growth rate reported by theoretical predictions. Stretching proved to be a process with good repeatability and able to reduce S. Typhimurium contamination by 5.5 Log CFU/g. The intrinsic characteristics of traditional WBMC proved to be unable to obstacolate the growth of S. Typhimurium during storage in the case of thermal abuse. Control of raw milk contamination and a proper refrigeration temperature are key factors in reducing the risk for consumers.

IDENTIFICATION OF VOLATILE COMPOUNDS, FREE AMINO ACIDS THROUGH CHROMATOGRAPHY AND SENSORY PROPERTIES OF THE PASTA FILATA CHEESES KNOWN AS MOMPOSINO

The cheese known as Momposino is a Colombian variety of pasta filata cheese. For this investigation, several cheese-production technologies were identified as follows: techniques 1 and 2, for cheeses elaborated from raw milk (such as autochthonous cheeses): technique 3, for cheeses made with pasteurized milk and with a culture; and technique 4, for cheeses made with raw milk. Volatile compounds and free amino acids were examined through chromatography and sensory evaluation tests performed by the members of laboratory. Cheeses made with techniques 1 and 2 presented more volatile compounds (with 60 and 73 respectively) than the cheeses made with technique 3 (for which 52 volatile compounds were found), and the ones made with technique 4 (which presented 40 volatile compounds). The fat content for cheeses from Technique 1 was 29.8%, 30% for cheeses from Technique 2, 27% for cheeses from Technique 3, and 25% for the ones made with Technique 4. These results lead to conclude that the higher the fat content is, the higher the volatile compound production will be. The volatile profiles that were found include acids, alcohols, hydrocarbons and esters. Through the Thin Layer Chrograthography test, it was found that cheese proteins spread to the following amino acids: arginine, valine, tryptophan, histidine, threonine, lysine, tyrosine, alanine, and cysteine. In the cheeses from all treatments, the most outstanding flavor was the acid flavor, and in the most outstanding texture was the elastic texture. No significant differences were found in the sensory evaluation, for the free amino acids (p>0.05), but free fatty acids and fat did show significant differences (p

Uncommonly Thorough Hydrolysis of Peptides during Ripening of Ragusano Cheese Revealed by Tandem Mass Spectrometry

Ragusano is a pasta filata cheese produced from raw milk in Sicily. The proteolysis was extensively analyzed after stretching (day 0), at 4 and 7 months of ripening through soluble nitrogen, urea-PAGE, and peptide identification by tandem mass spectrometry. After stretching, 123 peptides were identified: 72 arising from β-casein, 34 from α(s1)-casein, and 17 from α(s2)-casein. The main protein splitting corresponded to the action of plasmin, chymosin, cathepsin D, cell envelope proteinase, and peptidase activities of lactic acid bacteria. Unlike other types of cheeses, <10% residual β- and α(s)-caseins remained intact at 7 months, indicating original network organization based on large casein fragments. The number of identified soluble peptides also dramatically decreased after 4 and 7 months of ripening, to 47 and 25, respectively. Among them, bioactive peptides were found, that is, mineral carrier, antihypertensive, and immunomodulating peptides and phosphopeptides.

The effect of varying fat content on the microstructure of Oaxaca cheese, a typical pasta filata cheese

The effect of varying fat content in Oaxaca cheese, a typical pasta filata, on microstructure was described. Microstructure of cheeses was analysed by scanning electron microscopy (SEM) and light microscopy (LM) in nondehydrated and dehydrated samples. In nondehydrated samples, protein fibres were wide and compact in fat‐free cheese, and big serum channels were approximately 100 μm in width. Width of protein fibres and size of channels decreased as fat content increased. Small channels seemed to be occupied only by fat, while in big channels, water and fat were observed. LM both confirmed and supplemented the observations made by SEM, particularly the presence and distribution of fat in channels.

Effect of milk pasteurization and acidification method on the chemical composition and microstructure of a Mexican pasta filata cheese

Four Mexican pasta filata cheese variations were prepared using raw milk and starter culture (RMSC), raw milk and acetic acid (RMAA), pasteurized milk and starter culture (PMSC), and pasteurized milk and acetic acid (PMAA). Chemical composition, yield and microstructure were evaluated. Scanning electron micrographs of the cheese variations were evaluated by lacunarity analysis. PMSC and PMAA cheeses had higher protein, moisture and yield, but lower calcium content than RMSC and RMAA cheeses. The manufacturing conditions and refrigerated storage produced structural differences in the cheese variations, which displayed different heterogeneity patterns arising from spatial distribution of their components as determined by lacunarity analysis. Raw and pasteurized milk cheese variations showed an opposite lacunarity pattern trend during storage time.

Free fatty acids and other volatile compounds for the characterisation of “Vastedda della valle del Belìce” cheeseAcidos grasos libres y otros constituyentes volátiles para la caracterización de queso “Vastedda della vella del Belìce”

The analysis of the volatile constituents of “Vastedda della valle del Belìce”, a typical Sicilian pasta filata cheese, was performed using solid phase microextraction and high-resolution gas chromatography/mass spectrometry. The research aimed to verify if the volatile fraction, determinant for cheese flavor, differs among producers and/or production seasons. The samples were provided by four producers from the area of the Protected Designation of Origin (PDO) production, during two different seasons of production. A total of 42 volatile components were identified: the main components were found to be butanoic, hexanoic, octanoic, and decanoic acid. Free fatty acids were quantified using the standard addition method, their concentrations were in the range 1273.7–1918.0 mg/kg. The analysis of variance and a multivariate approach showed that producer and season factors significantly influenced the content of almost all the identified volatile components; the artisanal cheese-making, the utilization of raw milk, and the environmental differences that characterized the various farmers are strong causes of cheeses variability. El análisis de los constituyentes volátiles de ‘Vastedda della valle del Belìce’, un queso de pasta hilada (pasta filata) típica siciliana, fue llevado a cabo usando Microextracción en Fase Sólida y Cromatografía de Gases/Espectrometría de Masa de Alta Resolución. El objetivo de la investigación fue verificar si la fracción volátil, determinante para el sabor del queso, difiere entre productores y/o temporadas de producción. Las muestras fueron proporcionadas por cuatro productores del área de la Denominación de Origen Protegida (DOP) durante dos temporadas de producción diferentes. Se identificaron un total de cuarenta y dos componentes volátiles: los principales componentes encontrados fueron ácido butanoico, hexanoico, octanoico y decanoico. Los ácidos grasos libres fueron cuantificados usando el método de adición estándar, su concentración se encontró en el intervalo de 1273, 7–1918,0 mg/kg. El análisis de varianza y un a aproximación multivariada mostraron que productor y temporada influyeron significativamente en el contenido de casi todos los componentes volátiles identificados; la elaboración artesanal de queso, la utilización de leche cruda y las diferencias ambientales que caracterizaban a los diferentes productores son la causa de la variabilidad de los quesos.

CHARACTERIZATION OF LACTIC ACID BACTERIA ISOLATED FROM A TRADITIONAL PASTA FILATA CHEESE

In this paper are reported the results of the characterization of the lactic acid microflora isolated from Sardinian &ldquo;provoletta&rdquo;, a traditional pasta filata cheese obtained from cow&rsquo;s milk. Cheese samples from two batches were taken in triplicate from three dairy plants after 7 and 21 days of ripening. Each sample was analyzed for mesophilic and thermophilic lactic acid bacteria (LAB). From plates, 118 isolates were identified by Rep- PCR GTG5, species-specific PCR and DNA sequencing. The identified LAB belonged to 7 different species: Lb. paracasei, Lb. rhamnosus, Lb. delbrueckii, Lb. fermentum, E. faecalis, E. faecium e Lc. Lactis. Enterococci were the most common isolates, they were recovered from all the dairy plants and from all the products analysed at 7 and at 20 days of ripening. Although with some differences within the various producers, the technology used aids mesophilic LAB growth, guaranteeing a large biodiversity protection.

Short communication: Identification and technological characterization of yeast strains isolated from samples of water buffalo Mozzarella cheese

Sixty yeast cultures were isolated from samples of water buffalo Mozzarella, a popular “pasta filata” cheese, originating on 16 farms located in the provinces of Salerno, Caserta, and Frosinone (Italy). Strains were identified by means of 5.8S internal transcribed spacer rDNA PCR-RFLP combined with 26S rRNA gene partial sequencing and characterized for their ability to exert biochemical properties of technological interest. The recorded dominance of fermenting yeasts such as the lactose-fermenting Kluyveromyces marxianus (38.3% of the total isolates) and the galactose-fermenting Saccharomyces cerevisiae (21.6% of the total isolates) suggests that these yeasts contribute to the organoleptic definition of the water buffalo Mozzarella. The speciographic analysis revealed the presence of 7 other species rarely or never reported in a dairy environment belonging to the genera Pichia and Candida, whose role in Mozzarella cheese organoleptic properties need to be further investigated.

Oaxaca cheese: Manufacture process and physicochemical characteristics

Oaxaca cheese is a typical Mexican product of the pasta filata group. In spite of the importance of this cheese, it has been scarcely studied. The objective of this study was to document the method of manufacture and its physicochemical characteristics. There are variations in the process of manufacturing and has been affected by external influences such as time and temperature. There were variations in the physicochemical characteristics as the processes were not standardised, the variations being in protein from 150.3 to 241.5 g/kg; in fat from 170 to 253.3 g/kg; in pH from 4.9 to 5.8; in salt from 8.4 to 36.2 g/kg; in moisture from 124.1 to 610.3 g/kg and in ashes from 18 to 41.8 g/kg.

CHARACTERIZATION OF “SA PANEDDA” TRADITIONAL PASTA FILATA CHEESE OF MONTE ACUTO

Panedda is a traditional Sardinian food product. It is fresh pasta filata cheese made from cow’s milk, either raw or heat treated. The aim of this work was to acquire data on traditional cheesemaking process and microbiological profile of Panedda. In 3 dairies located in Monte Acuto area samples from 2 batches were collected during the whole cheesmaking process. Samples of raw milk, curd, acidified curd, pasta filata, and finished product after 7 and 20 days of ripening were collected and microbiologically analyzed. The results showed good hygienic quality in the finished product at 7 days and a decrease of coliforms, E. coli and coagulase-positive Stafilococci was observed during the further ripening. No pathogenic microorganisms were found in all the samples. Among Lactic Acid Bacteria rod-shaped forms were the most prevalent microflora and Enterococci reached levels of about 6 log cfu/g. The results of this study showed satisfactory hygienic characteristics of Panedda and will be useful to preserve traditional cheesemaking process.

Effect of freezing and frozen storage on microstructure of Mozzarella and pizza cheeses

Scanning electron microscopy was used to assess the effect of aging before (2, 7, and 14 days at 7 °C) or tempering after (1, 7, and 14 days at 7 °C) freezing, and frozen storage (1 and 4 weeks at −20 °C) on protein matrix of pasta filata Mozzarella and non-pasta filata pizza cheeses using unfrozen samples as controls. Pores and ruptures of reticular structure were observed in frozen-stored pasta filata Mozzarella cheese protein matrix, but cracks and clumps of bacteria were found in frozen-stored non-pasta filata pizza cheese. No obvious differences were discernable between the microstructures of pasta filata Mozzarella cheeses frozen stored 1 and 4 weeks. Formation of the reticular structure in frozen-stored pasta filata Mozzarella cheese progressed during tempering. Microstructure of non-pasta filata pizza cheese frozen stored for 4 weeks contained more extensive cracking and more areas of clumps of bacteria than that was frozen stored for 1 week. Aging of cheese before frozen storage was considered responsible for microstructural cracking; fewer cracks were found in the frozen-stored cheese tempered 1 and 2 weeks, but the clumps of bacteria were still observed.