The study on the effect of packaging methods on the shelf-life of iron fortified mozzarella cheese was undertaken in the Department of Livestock Products Technology and All India Coordinated Research Project on Post Harvest Engineering Technology, College of Veterinary Science, Assam Agricultural University, Khanapara, Guwahati during the period from August, 2016 to December, 2018.
Preparation of iron fortified mozzarella cheese
Iron fortified Mozzarella cheese was prepared from cow’s milk (100 per cent), goat’s milk (100 per cent) and mixed cow and goat milk (50:50) using chymax powder (control group) and kiwifruit crude extract (treatment group) as per standard technique laid down by Kanawjia
et al., (2011) with slight modification. Immediately after procurement, the milk was subjected to filtration followed by standardization to 3-4% fat level. The standardized milk was heated at 72°C/ 2min followed by cooling to 4°C. CaCl
2 was added @ 0.02g 100ml
-1 prior to acidification. Acidification was accomplished by the addition of 20% (w/v) citric acid to pH of 5.2–5.4 followed by addition of FeCl
3.6H
20 @1g/L. The temperature of the acidified milk was subsequently raised to 29°C and CHY-MAX Powder Extract NB was added @80 International Milk Clotting Unit/L of milk (control). For treatment groups, kiwifruit extract was used @150 µg/ml
. The milk in both the group was allowed to set until complete coagulation of the milk could be observed. The curd mass was then cut with sterile cheese knives and allowed to stand undisturbed in whey for 5min. Slowly the temperature of the curd was raised from 29°C to 38°C within 45min along with continuous stirring of the coagulated mass for uniform cooking and then held at 38°C for 30min. Whey was drained off and the curd mass was collected and stretched in hot water (85°C/10min) and molded into a ball. The cheese balls were then immersed in 20% w/v chilled brine solution for 2h followed by surface drying under refrigerated condition (7-9°C/6h). The prepared cheese samples were packed under two different packaging conditions, aerobically in polyethylene pouches and vacuum packed in high density polyethylene (HDPE) films of 200gauge (12×10²) packaged in Sevana
TM (Electrical Appliances Pvt. Ltd., Kerala, India) at a vacuum pressure of 720mm Hg and stored at refrigeration temperature throughout the entire study period.
Enumeration of microorganisms
Methods described by Harrigan and McCance (1976) were followed for enumerating the total viable count,
Escherichia coli, Salmonella, Shigella, Staphylococcus aureus, yeast and moulds and spores in prepared cheese samples by using appropriate media and incubation conditions.
Total viable count
One millilitre of appropriate dilution was transferred in to sterilized petri dishes in duplicates. Twenty millilitres of pre-sterilized, molten and cooled Plate Count Agar (PCA, Hi-Media, India) medium was poured into the petri dishes. The inoculum was mixed thoroughly by rotating the plates several times in clockwise and anti-clockwise directions. After solidification of the agar, the plates were incubated at 37°C for 24h. Colonies grown were counted and the counts were expressed as log
10cfu/g of sample.
Coliform count
Coliform count of the iron fortified mozzarella cheese were enumerated by inoculating 1ml of the diluent in Endo agar (Hi-Media, India) followed by incubation at 37°C for 24h. Average number of colonies counted was then expressed as presence or absence of coliforms in 0.1g of cheese samples.
Escherichia coli
E. coli count of iron fortified Mozzarella cheese was enumerated by inoculating 0.1g of the sample in Endo agar followed by incubation at 42°C for 24h. Results were interpreted as presence or absence of
E. coli in 1g of sample.
Listeria monocytogenes
Presence or absence of
Listeria monocytogenes of the iron fortified cheese sample was done by inoculating the required dilutions in Polymyxin-Acriflavin-Lithium Chloride-Ceftazidime-Aesculin-Mannitol (PALCAM, Hi-Media, India) agar and incubated at 37°C for 24h. Results were interpreted as presence or absence of
Listeria monocytogenes in 25g of sample.
Staphylococcus aureus
For detecting the presence of
Staph. aureus 1ml of appropriate dilution was transferred into sterilized Petri dishes in duplicates, in which 15-20ml of pre-sterilized and molten Mannitol Salt Agar (MSA, Hi-Media, India) medium was poured. The inoculum was mixed thoroughly by rotating the plates several times in clockwise and anti-clockwise directions. After solidification of the agar, the plates were incubated at 37°C for 24h. Results were inferred as presence or absence of
Staph. aureus in 1g of cheese sample.
Salmonella and Shigella
Twenty five grams of iron fortified mozzarella cheese samples were resuscitated in 100ml of sterilized nutrient broth and incubated at 37°C for 24h. The resuscitated sample was streaked on previously poured, solidified and sterility checked Salmonella-Shigella Agar (SSA, Hi-Media, India) plates and incubated at 37°C/24-72h. Results were interpreted as presence or absence of
Salmonella and
Shigella in 25g of cheese samples
.
Yeasts and moulds count
Yeast and moulds counts of the sample was done by inoculating the required dilutions in Rose Bengal Chloramphenicol Agar (RBCA, Hi-Media, India) and incubated at 37°C for 72-120h or till the formation of visible colonies. Results were interpreted as presence or absence of yeast and moulds in 1g of sample.
Anaerobic spore count
Anaerobic spore count of the samples was done by streaking from appropriate dilution on a previously poured, sterilized, sterility checked, solidified Starch Milk Agar (SMA) and incubated anaerobically at 37°C/24h. Results were inferred as presence or absence of anaerobic spores in 1g of cheese samples.
Statistical analysis
The experiment was conducted in a factorial RBD, considering 2 enzymes and 3 types of milk. Two-way ANOVA was performed in the generalized linear model (Proc GLM) as mentioned below and pair-wise comparison between the means was done by following the method of least significant difference (LSD) with the help of SAS 9.3.
Yijk = µ + Ei + Mj +ExMij + eijk
Where,
Y
ijk = Dependent variable (k
th observation in i
th enzyme and j
th milk).
µ = General effect.
E
i = Effect due to ith enzyme (i=1,2).
M
j = Effect due to jth milk (i=1,2,3).
ExM
ij = Interaction effect due to i
th enzyme and j
th milk.
e
ijk = Effect due to non-assignable causes.
