The Microbial Conversion Of Milk Into Cheese Biology Essay

Milk acts as an ideal civilization for assorted micro-organisms and bacteriums since it is really rich in all the indispensable constituents required for a growing medium. Its composing include C & A ; nitrogen beginnings, minerals, vitamins, enzymes, hint elements etc and besides favorable pH for growing of assortment of microorganisms & A ; bacteriums. ( Walker, G. and White, N. 2009 pers. Comm. , 16 November )

Traditionally milk has been used to bring forth butter, cheese yogurt etc but with the innovation of newer biotechnological techniques, it has been possible to bring forth assortment of merchandises like SCP ( individual cell protein ) , lactic acid, acetum, butyl alcohol, propanone, vitamins, antibiotics, ethanol etc.

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( Walker, G. and White, N. 2009 pers. Comm. , 16 November )

Cheese production in the universe is increasing significantly twenty-four hours by twenty-four hours. And the byproduct called whey which is produced during cheese production is besides increasing in big sums. So, processing of this whey into other utile merchandises must be done. Hence whey agitation is an of import facet in the dairy industry for bring forthing some merchandises like acetum ( Acetic Acid ) , ethanol etc which have a high commercial importance. The agitation procedure can be carried out utilizing barm species called Kluyveromyces marxianus ( barm used in big scale production of drinkable intoxicant from whey ) . This type of barm has an ability to change over lactose present in whey in to ethanol, C dioxide and barm biomass through agitation procedure.

( Walker, G. and White, N. 2009 pers. Comm. , 16 November )

Since milk Acts of the Apostless as a all right medium for growing of assorted bacteriums, micro-organisms it is really of import to cognize about types of microorganisms nowadays in milk, control methods and their benefits for worlds. Therefore there is demand for a microbiological analysis of the milk. Actually milk is unfertile when it is in the bag, but it is contaminated during milking, managing, storage, processing. One of the good bacterium nowadays in milk is lactic acid bacteriums. Whereas there can be assorted spoilage and infective micro-organisms like species of B, clostridia, cornebacterium, arthobacter, microbacterium, streptococci, pseudomonas etc.

In the research lab Sessionss, production of acetum and barm infusion was non done in a commercialised procedure but it has been merely designed to show assorted characteristics of agitation & A ; bioprocess engineering.

( Walker, G. and White, N. 2009 pers. Comm. , 16 November )

The chief aims of this research lab are:

Understand and analyze the existent industrial production of cheese in research lab itself.

Work expeditiously in groups i.e. , successful teamwork.

Exposure to assorted characteristics of agitation engineering.

To understand how a individual natural stuff ( milk ) could be converted to assorted commercially of import merchandises ( cheese, acetum, yeast infusion ) utilizing simple agitation techniques.

To derive in deepness cognition in the country of dairy biotechnology, a really large and of import nutrient based industry

Materials REQUIRED

1 milliliter, 10 milliliter pipettes, Petri dishes, conelike flasks, trial tubings, settlement counter, weighing machine, glass beads, Bunsen burner, vaccination cringle, microscope, yeast extract milk agar, VRBA ( violet red bile agar ) etc.


Actually the whole experimental procedure was carried out in three research lab Sessionss in which we were divided into three groups viz. :

Procedure squad ( does all the procedure related work )

Microbiology squad ( does all the microbiology related analysis assorted samples )

Analytic squad ( does analysis of assorted constituents from samples )

We were divided into groups because it helps in a smooth and organized manner of the procedure and therefore advancing squad work in the pupils. I was happened to be a member in the microbiology squad and hence we dealt with merely the microbiological analysis of milk, cheese and curd.


The natural milk was inoculated with lactic starter civilization ( streptococcus cremoris/lactobacillus lactis ) , Annatto ( ruddy coloring compound ) and besides Rennet ( obtained from calf bowel ) for formation of clot. This clot was cut into pieces and so cooked for certain clip to acquire curd and whey. Then whey was separated from the curd. And curd was processed into cheese through salting, organizing & A ; maturating. The detached whey was subjected to extremist filtration, which resulted in two constituents:

Whey permeate

Whey retentate

Whey retentate can be farther processed to obtain whey proteins which are used extensively in nutrient material. Further some barm inoculant was added to the whey permeate to originate the procedure of agitation. This was done with two samples A and B ( in which extra milk sugar was added externally ) . The agitation was carried out for 5 yearss and for every one twenty-four hours the samples were collected and kept aside for the analysis of lactose and protein. After the agitation, both samples were subjected to centrifugation. This consequences in ethyl alcohol ( whey vino ) and besides yeast biomass ( pellet ) . The yeast biomass was further processed to obtain yeast infusion, a nutrient additive with broad scope of applications in nutrient industry. The ethyl alcohol was farther subjected to acetification, which consequences in formation of acetic acid utilizing Acetic acid bacterium ( Acetobacter aceti ) . This Acetic acid was clarified utilizing wood coal column to acquire acetum. ( Walker, G. and White, N. 2009 pers. Comm. , 16 November )



For this method, the suggested medium was Yeast extract milk agar ( a medium which selectively supports the growing of micro-organisms associated with milk & A ; its merchandises ) .

A series of consecutive dilutions of the milk sample were made like:

1 in 10 dilution = 9 ml H2O + 1 milliliters milk

1 in 100 dilution = 9 ml H2O + 1 milliliter of 1 in 10 dilution

1 in 1000 dilution = 9 ml H2O + 1 milliliter of 1 in 100 dilution

During these dilutions at every measure each trial tubing was assorted to do the solution uniform. Harmonizing to the Scots ordinances, merely 1 in 100 and 1 in 1000 dilution samples were tested. 1 milliliter of 1 in 100 and 1 milliliter of 1 in 1000 dilution samples were placed in two separate Petri dishes and afterwards 10 milliliter of malted agar was poured into the Petri dish. After the hardening of medium, the home bases were incubated in upside-down place for 72 hours at 30 grades centigrade. After the incubation, the figure of settlements in each home base were counted utilizing settlement counter and it was so multiplied by the dilution, and the end point is called aa‚¬A“number of feasible bacterial per mlaa‚¬A? .


In this method, the medium used was VRBA ( Violet Red Bile Agar ) , a medium used for numbering of coliform beings in dairy merchandises. It has high selectivity towards coliforms due to the presence of gall salts and crystal violet.

The consecutive dilutions were done as for the first method. Then utilizing overlay method, foremost pour 1 milliliter of 100 and 1ml of 1 in 1000 dilutions in Petri home bases and so 8 milliliters of fresh molten VRBA was poured. It was allowed to put and eventually it was overlaid with 3-4 milliliter of fresh molten VRBA.

Overlay method ensures anaerobiotic conditions which suppress growing of non fermentative gm negative bacteriums. It besides encourages the lactose agitation which finally favours formation of seeable violet settlements.

The Petri home bases were incubated at 37 grades centigrade for 72 hours. After the incubation, the Petri home bases were used to number the coliforms which were present in the seeable violet coloring material settlements. Coliform count was done as settlement organizing units ( CFU ) per milliliter milk.


The cheese used for microbiological analysis was old one, which is six months old cheese. And the curd which is formed during the cheese production procedure was used for the microbiological analysis intent. The analysis for both of them was done same as that of milk, but with some minor alterations. Here alternatively of 1 milliliters of milk, 1 gm of curd every bit good as cheese were used for the consecutive dilution intent. Besides, the 9 milliliter clean solution was added with some glass beads. Besides the dilution for cheese and curd were 1 in 100, 1 in 1000, and 1 in 10000. And similar to the milk, general feasible counts and entire coliform counts were performed.


Table 1: Calculations for the feasible count of micro-organism in tried samples.

Tested Samples


10-2 10-3 10-4

Average of settlement count ( cfu/ml )


1.0 x 103cfu/ml

7.0 x 103cfu/ml


8.0 x 103


1.52 x 104cfu/ml

1.12 ten 105 cfu/ml

7.8 x 105cfu/ml

9.0 x 105

Old Cheese

7.0 x 102cfu/ml

5.3 x 104cfu/ml

3.0 x 105cfu/ml

3.54 x 105

Average feasible settlement count for milk was calculated as 8.0 x 103 CFU/ml

Average feasible settlement count for curd was calculated as 9.0 x 105 CFU/ml

Average feasible settlement count for old cheese was calculated as 3.54 x 105 CFU/ml

Table 2: Calculations for the coli signifier count in tried samples.

Tested Samples


10-2 10-3 10-4

Average of settlement count ( cfu/ml )


5.0 x 103cfu/ml

2.4 x 104cfu/ml


2.9 x 104


1.56 ten 104 cfu/ml

9.5 x 104cfu/ml

6.3 ten 105 cfu/ml

7.4 x 105

Old Cheese

7.6 x 103cfu/ml

1.01 x 105cfu/ml

6.1 x 105cfu/ml

7.19 x 105

Average coli signifier count for milk was calculated as 2.9 x 104 CFU/ml

Average coli signifier count for curd was calculated as 7.4 x 105 CFU/ml

Average coli signifier count for old cheese was calculated as 7.19 x 105 CFU/ml

CFU ( settlement organizing units )

It is one of the unit of measurings used in microbiology which indicates the figure of microorganisms nowadays in 1 milliliter of the sample.

Bacterial taint was besides reported in 2 of the milk sample home bases ( 1 in 100 dilution & A ; 1 in 1,000 dilution ) .

Contaminants in milk sample home base of 1 in 100 dilution: one out of two major contaminations had features like gm positive nature and cells were present in extended. The other contaminations which were present on the peripheral part were gram positive, rod shaped and unvarying in morphology. No extended cells were present with regard to the ulterior contaminations.

Contaminants in milk sample home base of 1 in 1000 dilution: the contamination showed the gm positive nature and microscopic scrutiny revealed that the cells were in the signifier of rod shaped and some of the cells still attached to each other.


Figure 1: appraisal of lactose concentration utilizing DNS methods

Figure 2: Lactose Concentration and OD of Milk, Whey Permeate and Retentate

Figure 3: Analysis of lactose and ethanol concentration with barm cell count in sample A w.r.t clip

Figure 4: Analysis of lactose and ethanol concentration with barm cell count in sample B w.r.t clip

Figure 5: Analysis of ethyl alcohol and entire acid in sample Angstrom

Figure 6: Analysis of ethyl alcohol and entire acid in sample B

Figure 7: appraisal of protein concentration in milk, whey permeate, whey retentate



Vial # 1 = 6.471 % ethyl alcohol ( v/v ) Vial # 1 = 1.819 %

Vial # 2 = 5.376 % Vial # 2 = 1.449 %

Vial # 3 = 6.319 % Vial # 3 = 0.580 %

Vial # 4 = 3.394 % Vial # 4 = 0.085 %

Vial # 5 = 3.003 % Vial # 5 = ND ( non

detected )


Harmonizing to the Scots ordinances there must be less than15000 bacteriums per milliliter in the premium milk, and with the bacterial count obtained in our analysis were around 8000 bacteriums per milliliter of milk. This clearly shows that the milk is free from bacterial taint, but there is another status to be satisfied i.e. there must be no coli signifier bacteriums in 1 in 100 and 1 in 1000 dilution milk samples. But unluckily might be due to hapless handling conditions, we observed taint in both the samples. After recognizing that there was taint in the samples, the polluting bacterium was analysed under the microscope utilizing the gramaa‚¬a„?s staining. We discovered that the beings that contaminated the samples were both gram positive, rod shaped beings. One of them was suspected to be a species of Bacillus coagulans, a lactic acid organizing bacteriums and besides acts a probiotic. And the other one was like assorted proteolytic bacteriums with an antibiotic sort of activity environing it in the Petri dish. One more observation that was made is the figure of bacterial count in curd was higher than that of milk and besides old cheese indicating that more figure of beings are survived in curd, may be most of them good to worlds! !


The experiment proved that milk can be successfully converted into cheese, acetum and barm infusion. Cheap whey was converted into expensive acetum and ethyl alcohol and it is really fresh and industrially feasible procedure. Besides attending could farther be paid towards betterment of more efficient barm strains which can utilize low milk sugar degrees to give maximal end product. Dividing the pupils into groups for transporting out assorted subdivisions of the experiment was really good and therefore encourages team work in them. Overall the whole procedure carried out was good but if the pupils were taken to some industry, they can acquire an existent image of how the procedure is done in industrial graduated table and helps in better apprehension of this lab and adds meaningfulness to it.