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Matteo Alessandro Del Nobile
Influence of the milk bactofugation and natural whey civilization on the microbiological and physico-chemical features of mozzarella cheese
Michele Faccia1, Marianna Mastromatteo2, Amalia Conte2, Matteo Alessandro Del Nobile2
1 Department of Soil, Plant and Food Science, University of Bari, Via Amendola 165/A 70126 Bari, Italy
2 Department of Agriculture, Food and Environment Science, University of Foggia, Via Napoli, 25 71122 Foggia, Italy
Matching Writer: Del Nobile M.
Complete full name: Matteo Alessandro Del Nobile
Complete get offing reference: Department of Agriculture, Food and Environment Science, University of Foggia, Via Napoli, 25 71100 – Foggia, Italy
Facsimile: ( +39 ) 881 589 242
Telephone: ( +39 ) 881 589 242
Electronic mail: ma.delnobile @ unifg.it
In this work the consequence of the milk bactofugation and the natural whey civilization on the microbiological and physico-chemical quality of mozzarella cheese was studied. To this purpose the microbiological, centripetal and physico-chemical parametric quantities of the mozzarella cheese were monitored during the storage at 8 C. The bactofugation intervention did non impact significantly the growing of typical dairy micro-organisms while a important lessening of Enterobacteriaceae in milk was found. The mozzarella manufactured with natural whey civilization and bactofugated milk showed a slower addition in the Pseudomonas spp. cell burden during storage. Furthermore, the absence of natural whey civilization in the mozzarella cheese industry caused a faster sensory quality loss during storage ( ? 4.5 yearss ) respect to the merchandise with natural starting motor ( ? 6 yearss ) . However, the factor restricting the shelf life of mozzarella cheese was the growing of Pseudomonas spp. In peculiar, a shelf life value of about 4.0 yearss for mozzarella produced with bactofugated milk and natural whey civilization and 3.5 yearss for the control, with citric acidification, and samples manufactured with natural whey civilization and no bactofugated milk was obtained. The compositional features of the cheeses were influenced by the usage of the natural starting motor, specially at the terminal of the storage period. In fact, the control cheese tended to loss faster the soluble compounds ( WSN and NaCl ) and to increase the wet content.
Keywords: mozzarella cheese, bactofugation, natural whey civilization, shelf life.
The pasta filata procedure of cookery and stretching of cheese curd is typical during industry of many Italian cheeses such as the Mozzarella cheese [ 1 ] . Two types of Mozzarella are produced, based on its wet content [ 2 ] . In peculiar, low wet ( LM ) Mozzarella cheese ( moisture 45-52 % ) is normally used for pizza toppings or as an ingredient in other formulas, whereas high wet ( HM ) Mozzarella cheese ( moisture 52-60 % ) , doing up most of the Italian Mozzarella export, is normally consumed fresh as a tabular array cheese [ 3,4,5 ] . Traditional HM mozzarella is packaged in a dilute salt solution ( NaCl and/or CaCl2 ) ( called conditioning seawater ) and has a short shelf life ( 34 yearss ) . The short shelf life of traditional mozzarella cheese has been attributed to microbiological spoilage. This spoilage is frequently caused by the growing of coliforms, Pseudomonas spp. and/or by psychrotrophic bacteriums that grow on the cheese surface, largely coming from H2O used in the industry [ 6,7,8 ] .
Under industrial conditions, selected thermophilic lactic acid bacterium civilizations are used as starting motors in Mozzarella cheese doing [ 9 ] , whereas for traditional processing, of course fermented whey is used as the inoculant. The choice of starting motor civilizations has a profound consequence on the concluding features of the cheese. Yun et Al. [ 10 ] reported that an appropriate rod-to-coccus ratio influences the proteolysis, give and viscousness of Mozzarella cheese. In the traditional cheese devising procedures the natural whey civilizations are natural microbic civilizations happening in the whey drained after curd maturation. Part of this whey is stored and employed as starting motor in the industry of the undermentioned twenty-four hours. However, small information is available on the complex bacterial community of Mozzarella cheese from natural cattles milk processed by adding the of course fermented whey from the old twenty-four hours as a starter civilization [ 11,12 ] .
Bactofugation is a procedure in which a specially designed hermetic extractor, the Bactofuge, is used to divide bacteriums, and particularly the spores formed by specific bacteriums strains, from milk. Bactofugation has proved to be an efficient manner of cut downing the figure of spores in milk, since their denseness is higher than that of milk. Bactofugation usually separates the milk into a fraction that is more or less free from bacteriums, and a dressed ore ( bactofugate ) , which contains both spores and bacteriums in general and sums to up to 3 % of the provender to the Bactofuge. It has chiefly been used in the cheese industry where its high-cleaning capablenesss have been used to take spores from cheese milk that could do latent agitation in semi-hard cheeses. It has now been adapted for treating ingestion milk, where it holds out the promise of protracting the life of fresh, pasteurised milk by three-five yearss [ 13 ] . The consequence of assorted governments of milk bactofugation on the remotion of bacterium was studied by Dilanyan et Al. [ 14 ] . Bactofugation of either cold milk or of milk pre-heated to 43-45C eliminated about 67-85 % of the bacteriums present ; when dual intervention was carried out the bacterial remotion increased to approximately 95 % . Bactofugation can be an interesting option to pasteurisation in cheese-making: it allows to utilize natural milk, avoiding the negative effects of heating to the curdling belongingss [ 15 ] . This facet is peculiarly interesting for cheeses that require curd with good soundness and construction, such as difficult, semi-hard and pasta filata cheeses. For this latter type of cheese, equal curd construction and low presence of whey proteins are really of import to vouch good stretching belongingss [ 16 ] . For Italian Mozzarella the structural features are peculiarly of import for one more ground, that is the storage of the cheese in H2O or diluted seawater, which tends to impair soundness of the merchandise. Indeed, bactofugation is non comparable to pasteurisation as to safety, even though it is really effectual towards coliforms [ 17 ] : however, the presence of a thermal measure ( the stretching procedure ) during the industry, makes the bactofugation procedure compatible with the safety of the concluding merchandise. For all these grounds some dairies are re-considering bactofugation as an option to pasteurisation ; unluckily, information sing the application of this technique for the industry of Mozzarella is missing.
Taking into history all the above considerations, the aim of the present survey was to measure the consequence of the milk bactofugation and the natural whey civilization on the microbiological, centripetal and physico-chemical quality of HM mozzarella cheese.
2. Materials and Methods
The bactofugation procedure was carried out in a industrial dairy, utilizing the one-phase Westfalia bactofuge CNE 300 at the following operating conditions: temperature 55C ; flow rate 30.000 L/h ; feeding force per unit area 1 saloon ; force per unit area at discharge 5 saloon.
Mozzarella cheese industry
Samples of Mozzarella were produced from natural ( NWCno bacto ) or bactofuged milk ( NWCbacto ) , both inoculated with 5 % natural whey civilization obtained by self-generated agitation, at 38 C, of whey derived from the cheese production of the old twenty-four hours. Bovine rennet add-on ( strength 1:10.000 at the dose 0.25 mL L-1 of milk ) was performed at 35 C and curdling took topographic point in 20 min. After remotion of approximately 80 % whey the curd was kept warm for agitation, which was completed within 3 hours. Samples of Mozzarella were besides produced from the same natural milk by direct acidification with citric acid, utilizing the protocol reported in a prevoius paper [ 18 ] , and used as control ( CNTR ) . Curd stretching was performed automatically, utilizing hot H2O at 85 C. During the stretching, the temperature of the curd increased to 60 C. After determining, the cheeses were placed in cold H2O ( 10 C ) and stored at 4 C until trying.
The readying of the trial samples, initial suspension and denary dilutions for microbiological scrutiny was performed harmonizing to the International Standard ISO 8261:2001. Plate Count Agar ( PCA ) incubated at 30 C for 2448 H was used as media for entire feasible count ; Pseudomonas Agar Base ( PAB ) , added with CFC selective addendum, incubated at 25 C for 48 H for Pseudomonas spp. count. For Enterobacteriaceae, Violet Red Bile Glucose Agar ( VRBGA, Oxoid ) was used and home bases were incubated at 37 C for 1824 h. Lactic acid B ( LAB ) were plated on de Man Rogosa Sharpe agar ( MRS, Oxoid ) and incubated anaerobically in the jars HP 11 ( Oxoid, Milan, Italy ) at 30 C for 24 yearss. M17 agar ( Oxoid ) , incubated at 37 C for 48 H for coccus-shaped lactic acid bacteriums. For the numbering of enterococci, Slanetz-bartley medium incubated at 37C for 48-72 H, was used. Sabouraud Dextrose Agar incubated at 25C for 48 H, was used as media for barm count. The microbiological analyses were carried out twice on two different batches.
In order to measure the consequence of the bactofugation on the microbiological quality of milk, Microbial Growth Decrease index was calculated as:
where log CFUc ml-1 represents the cell burden of control milk and log CFUb ml-1 the cell burden of bactofugated milk.
The microbic acceptableness bound ( MAL ) ( i.e. , the storage clip at which the viable cell concentration reached the threshold ) , was calculated as reported by Del Nobile et Al. ( 2009 ) . The bound of the cfu g-1 was fixed at 106 cfu/g for Pseudomonas spp. [ 19 ] .
Mozzarella cheese samples were analysed for: wet [ 20 ] , protein by macro-Kjeldahl method [ 21 ] , fat by Soxhlet method, pH [ 22 ] , water-soluble N ( WSN ) by the method of Kuchroo and Fox [ 23 ] . All compositional analyses were performed in triplicate. The profile of WSN was studied by High Performance Liquid Chromatography utilizing a Waters 990E pump, equipped with a VydacC18 column and a Diode Array Detector 996 ; the separation was carried out with a gradient of acetonitrile in H2O, both incorporating 0.1 % trifluoracetic acid, from 5 to 70 % in 45 min.
Mozzarella samples were subjected to centripetal analysis by a panel consisting of seven trained judges. Panelists of the nutrient packaging research lab of the University of Foggia were selected on the footing of international criterions ISO 8586-1:1993 ( Sensory analysis- General counsel for the choice, preparation and monitoring of assessors Part 1: Selected assessors ) and ISO 8586-2:1994 ( Sensory analysis- General counsel for the choice, preparation and monitoring of assessors Part 2: Experts ) . Mozzarella samples were presented to panellists without seawater and they were asked to depict differences between samples by utilizing a graduated table from 1 to 7 [ 24 ] . Panelists were asked to establish their determination on the sample overall quality merely taking into history colour, olfactory property, and soundness. Therefore, the samples overall quality has to be considered as an norm of the above-named sensory properties ( i.e. , colour, olfactory property, and firmness ) as weighted by the panellist [ 4 ] . A mark of 4 was the minimal threshold value for cheese acceptableness. The centripetal acceptableness bound ( SAL ) ( i.e. , the storage clip at which the sensory property reaches the threshold ) was calculated as reported by Del Nobile et Al. [ 4 ] .
Experimental informations of all tested samples were compared by one-way Anova analysis. A Duncans multiple scope trial, with the option of homogenous groups ( P & A ; lt ; 0.05 ) , was used to find significance among interventions. To this purpose, Statistica 7.1 for Windows ( StatSoft Inc. , Tulsa, OK, USA ) was used.
3. Consequences and Discussion
Consequence of bactofugation on milk microbic quality
The consequence of the bactofugation on the microbiological quality of milk is shown in figure 1. It has been found that, on the norm, milk treated in a bactofuge contains 90 % fewer sources than the original milk [ 25 ] . In our conditions, among the anaerobe-facultative micro-organism the highest cell burden lessening was found for Enterobacteriaceae ( about 72 % ) , in understanding with the consequences reported by Kosikowsky and Fox [ 17 ] . The bactofugation did non impact significantly the growing of typical dairy micro-organism. In peculiar, a low MGD index for enterococci ( 7 % ) , coccus-shaped LAB ( 26 % ) and lactic acid B ( LAB ) ( 33 % ) was obtained. Furthermore, the microbic growing lessening was approximately 10 % for entire feasible count and 55 % and 21 % for barm and Pseudomonas spp. , severally. These consequences are interesting for the Mozzarella cheese-making procedure, since they demonstrate that the conditions used for the bactofugation procedure allow to obtain a good choice of micro-organisms without utilizing high temperature. Furthermore, it is good known that the effectivity of bactofugation additions with the temperature of the milk.
The figure 2 shows the microbic cell burden of the natural whey civilizations ( NWC ) used in the cheese-making procedure. The natural whey civilizations consisted of a big figure of micro-organisms and a great assortment of microbic groups. The consequences showed a entire feasible count of about 6.0 log cfu/ml with a predomination of lactic acid bacteriums ( 6.0 log cfu/ml ) and enterococci ( 5.0 log cfu/ml ) . Their ability to bring forth spirit compounds and to synthetize big sums of extracellular polyoses could be of import in mozzarella cheese industry. Yeasts and coccus-shaped LAB reached counts in the scope of 4 log cfu/ml and they may be responsible for some biochemical activity in the cheese-making procedure. Enterobacteriaceae in the natural whey civilizations were below the sensing bound while a cell burden of about 4.5 log cfu/ml for Pseudomonas spp. was recorded. Natural whey civilizations were capable to variable storage temperatures under high acid conditions for extended periods. Therefore the happening of Pseudomonas spp. was likely due to the opposition at the damaging effects of high sourness.
Microbiological quality of mozzarella cheese
The development of Pseudomonas spp. cell load plotted as a map of storage clip for mozzarella samples is shown in Figure 3a. In the figure the horizontal solid line represents the feasible cell concentration threshold value ( 106 cfu/g ) . It is deserving observing that microbic cell burden was monitored until the packed nutrient reached either its microbial or centripetal threshold value. As can be seen the NWCbacto samples showed the lowest initial cell burden of Pseudomonas spp. ( below 2 log cfu/g ) . Regards the CNTR and the NWCno_bacto samples a cell burden of about 4 and 3 log cfu/g, severally, was observed. However, during the storage clip, Pseudomonas spp. cell burden of the latter two samples exceeded the threshold value at earlier storage clip if compared to try manufactured with NWC and bactofugated milk ( NWCbacto ) . In fact, the MALPseudomonas values listed in Table 1 high spot that the add-on of NWC and the usage of bactofugated milk ( NWCbacto ) significantly affected the microbic quality of mozzarella cheese ( P & A ; lt ; 0.05 ) . On the other manus, the NWCno_bacto samples showed non significant differences compared the mozzarella without NWC ( CNTR ) . For these samples a MALPseudomonas values of about 3.5 yearss was recorded ; whereas, for the NWCbacto samples a value of about 4.5 yearss was obtained.
Figure 3b shows the development of entire feasible count for mozzarella samples during the storage. As can be seen from informations shown in the figure, a faster addition of the entire feasible count was observed for CNTR sample. In peculiar, the initial cell burden was about 5.0 log cfu/g and reached a value higher than 8.0 log cfu/g at 6 yearss of storage. It is deserving observing that for the NWCbacto samples a slower growing regard to the NWCno_bacto samples was observed. Furthermore, a lower cell burden of about 0.65 log rhythm for NWCbacto samples at 7 yearss of storage was obtained.
Lactic acid B for NWCbacto samples showed a lower addition at 5 yearss of storage respect the other samples ( Fig. 3c ) . However, at the terminal of storage the same cell burden for all samples was observed.
It is deserving observing that for NWCbacto samples the coccus-shaped lactic acid bacterium showed a lowest addition regard to that from no bactofugated milk ( NWCno_bacto ) and CNTR ( Fig. 3d ) . In peculiar, a concluding cell burden of about 7.5 log cfu/g for CNTR and NWCno_bacto samples was recorded. A difference of about 1.0 log rhythm for NWCbacto samples was observed.
The enteric bacteria cell burden in the NWCbacto samples was below 4 log cfu/g until the fifth yearss and reached a cell burden of about 6 log cfu/g at the terminal of storage ( Fig. 3e ) . For mozzarella samples NWCno_bacto a faster addition of the Enterobacteriaceae cell burden was observed. The same development for the CNTR samples was besides obtained ; furthermore, in this instance, a higher initial ( 3.6 log cfu/g ) and concluding cell burden ( 7.3 log cfu/g ) was recorded.
The gross composing of the three types of mozzarella at 1 twenty-four hours were non statistically different as to moisture, fat, protein and NaCl, whereas pH and WSN showed some differences between the two samples made with natural starting motor and the control ( Table 1 ) . As expected, pH was higher in the control cheese due to the fact that it was produced by direct acidification. The WSN content was really low in all samples bespeaking really hapless proteolysis, as antecedently reported [ 26 ] , but it was peculiarly low in the control. The values of WSN decreased with clip: the informations at twenty-four hours 7, beside corroborating scarce casein debasement, suggests that a progressive loss of low molecular weight compounds ( likely migrating into the storage solution ) takes topographic point. This hypothesis is confirmed by the development of the concentration of sodium chloride: for all samples a lessening was found, but it was more pronounced for the control ( from 8 to 2.5 g kg-1 ) . For this sample, a little addition of the wet content was besides detected.
The HPLC profiles of WSN at twenty-four hours 7 are reported in fig. 4: they evidenced some differences. The profiles of the samples made natural starting motor were really similar under a qualitative point of position, but that of the No Bacto sample had somewhat higher extremum. The profile of the control was alternatively wholly different: the extremums were less high and were chiefly shifted towards the concluding portion of the chromatogram, bespeaking a more pronounced hydrophobicity.
Centripetal quality decay
Figure 5a shows the development during storage of the olfactory property in mozzarella cheese. The mark of the olfactory property faster decreased for the CNTR samples making foremost the olfactory property threshold value ( hit 4 ) . The panellists judged the samples manufactured with NWC with high olfactory property mark ; moreover, the bactofugation intervention did non impact this parametric quantity. Color property showed high mark in the first 4 yearss of storage and so decreased ; nevertheless, no difference between the samples was observed ( Fig. 5b ) . It is deserving observing that the texture was affected by both the add-on of NWC in the production procedure and the bactofugation intervention. In peculiar, the texture mark for the CNTR sample decreased if compared to the other investigated samples, making the threshold value at about 5 yearss, followed by the NWCno_bacto samples at 6 yearss. The bactofugation procedure positively affected the texture of the merchandise that reached the threshold value at 7 yearss ( Fig. 5c ) .
The overall quality decay of mozzarella samples is shown in Figure 5d. As can be seen, the overall quality tendency coincides with mark of the olfactory property, therefore turn outing that this property represented the factor restricting cheese storability. In peculiar, the overall quality of the CNTR samples, after the first three yearss, quickly decreased making foremost the overall quality threshold value ( hit 4 ) . The add-on of NWC during the industry procedure affected positively the sensory quality of mozzarella. In fact, the overall quality for this sample remained above the threshold value up to the 6th twenty-four hours. After this period the quality decay occurred. It should be noted besides that the bactofugation did non impact the sensory quality of produced mozzarella.
SAL values determined for mozzarella cheese overall quality ( SALOverall Quality ) were listed in Table 2. As declared beforehand, any significant difference between mozzarella manufactured with NWC and milk subjected or non to the bactofugation intervention was observed ( P & A ; gt ; 0.05 ) . On the other manus, the absence of NWC in the mozzarella cheese industry caused a faster sensory quality loss during storage. In peculiar, a SALOverall Quality value of about 6 and 4.5 yearss for mozzarella with ( NWCbacto, NWCno_bacto ) and without NWC ( CNTR ) was recorded.
Shelf life rating
Mozzarella shelf life was calculated as the lowest value between MALPseudomonas and SALOverall Quality ( Table 1 ) . It is deserving observing that the shelf life of mozzarella cheese seemed to be strongly dependent on the microbic quality. In peculiar, Pseudomonas spp. growing was the confining factor the merchandise acceptableness, whereas the sensory quality did non restrict the mozzarella shelf life. As can be inferred from informations, for CNTR and NWCno_bacto samples a shelf life value of about 3.5 yearss and 4.5 for the NWCbacto samples was obtained.
Consequences of this survey suggested that bactofugation can be successfully used to take the bacteriums of milk prior to treating. Therefore, the bactofugation procedure can be an interesting option to pasteurisation in cheese-making as it avoid the negative effects of heating to the curdling belongingss. Furthermore, this technique is able to better the shelf life of mozzarella obtained with natural milk with the add-on of natural whey civilization. In fact, for these samples a lower addition of the Pseudomonas spp. cell burden was obtained. Furthermore, the absence of natural whey civilization in the mozzarella cheese industry caused a faster sensory quality loss during storage regard to the merchandise with natural whey civilization. However, the factor restricting the shelf life of mozzarella cheese was the growing of Pseudomonas spp. , whereas the sensory quality did non restrict the merchandise acceptableness. In peculiar, a shelf life value of about 4.5 yearss for mozzarella with natural whey civilization and bactofugated milk and 3.5 yearss for the control and sample with natural whey civilization and no bactofugated milk was obtained. The compositional features of the cheeses were influenced by the usage of the natural starting motor, in peculiar at the terminal of the storage period, when the control cheese tended to loss faster the soluble compounds ( WSN and NaCl ) and to increase the wet content. These consequences suggest a more pronounced permeableness of the casein matrix made by direct acidification: the grounds of this different behaviour among the cheeses need more probe.
Caption TO FIGURES
Figure 1 Consequence of the bactofugation on milk microbiological quality.
Figure 2 Microbial cell burden of natural whey civilizations ( NWC ) used in the cheese-making procedure.
Figure 3 Development of Pseudomonas spp. ( a ) , entire feasible count ( B ) , lactic acid B ( degree Celsius ) , coccus-shaped lactic acid bacteriums ( vitamin D ) , Enterobacteriaceae ( vitamin E ) for mozzarella cheese during the storage.
Figure 4 HPLC profiles of WSN of the mozzarella samples.
Figure 5 Development of olfactory property ( a ) , colour ( B ) , texture ( degree Celsius ) and overall quality ( vitamin D ) for mozzarella cheese.
Table 1. Chemical composing of mozzarella samples during storage ( g kg-1 ) .
NWCbacto NWCno bacto CNTR
1d 7d 1d 7d 1d 7d
pH 5.350.01a 5.410.03b 5.310.04a 5.430.02b 5.860.03c 5.960.03d
Moisture 6206.0a 6265.5a 6154.0a 6215.5a 6165.5a 6395.0b
Fat 1554.5a, b 153.3.5 a, B 1561.0b 1545.0a, b 1583.0b 1513.0a
Protein 1482.0a, B 1494.0a, B 1492.5a, B 1482.0a, b 1512.0b 1433.5a
NaCl 7.40.3 degree Celsiuss 3.90.4 b 7.60.2c 3.80.2 B 8.00.5c 2.50.2 a
WSN 0.120.01c 0.100.03b, c 0.140.05c 0.110.02c 0.070.01b 0.040.01a
Valuess with different superiors are different at P & A ; lt ; 0.01.
Table 2. Shelf life of mozzarella samples evaluated on the footing of microbic acce1ptability bound ( MAL ) of Pseudomonas spp. ( MALPseudomonas ) and sensory acceptableness bounds ( SALOverall Quality ) .
Samples MALPseudomonas SALOverall Quality Shelf Life
CNTR 3.570.16a 4.480.23a 3.570.16a
NWCbacto 4.330.38b 6.330.18b 4.330.38b
NWCno bacto 3.400.20a 5.950.17b 3.400.20a
Meanss in the same column followed by different superior are significantly different ( P & A ; lt ; 0.05 ) .
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