The Microbial Conversion Of Milk Into Cheese Biology Essay

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Milk acts as an ideal culture for various microorganisms and bacteria since it is very rich in all the essential components required for a growth medium. Its composition include carbon & nitrogen sources, minerals, vitamins, enzymes, trace elements etc and also favourable pH for growth of variety of microorganisms & bacteria.(Walker, G. and White, N. 2009 pers. Comm., 16 November)

Traditionally milk has been used to produce butter, cheese yoghurt etc but with the invention of newer biotechnological techniques, it has been possible to produce variety of products like SCP (single cell protein), lactic acid, vinegar, butanol, acetone, vitamins, antibiotics, ethanol etc.

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

Cheese production in the world is increasing significantly day by day. And the by-product called whey which is produced during cheese production is also increasing in large amounts. So, processing of this whey into other useful products must be done. Hence whey fermentation is an important aspect in the dairy industry for producing some products like vinegar(Acetic Acid), ethanol etc which have a high commercial importance. The fermentation process can be carried out using yeast species called Kluyveromyces marxianus (yeast used in large scale production of potable alcohol from whey). This type of yeast has an ability to convert lactose present in whey in to ethanol, carbon dioxide and yeast biomass through fermentation process.

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

Since milk acts as a fine medium for growth of various bacteria, microorganisms it is very important to know about types of microorganisms present in milk, control methods and their benefits for humans. Therefore there is need for a microbiological analysis of the milk. Actually milk is sterile when it is in the udder, but it is contaminated during milking, handling, storage, processing. One of the beneficial bacteria present in milk is lactic acid bacteria. Whereas there can be various spoilage and pathogenic microorganisms like species of bacillus, clostridium, cornebacterium, arthobacter, microbacterium, streptococcus, pseudomonas etc.

In the laboratory sessions, production of vinegar and yeast extract was not done in a commercialized process but it has been only designed to demonstrate various features of fermentation & bioprocess technology.

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

The main objectives of this laboratory are:

Understand and analyse the actual industrial production of cheese in laboratory itself.

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

Exposure to various features of fermentation technology.

To understand how a single raw material (milk) could be converted to various commercially important products (cheese, vinegar, yeast extract) using simple fermentation techniques.

To gain in depth knowledge in the area of dairy biotechnology, a very big and important food based industry

MATERIALS REQUIRED

1 ml, 10 ml pipettes, Petri dishes, conical flasks, test tubes, colony counter, weighing machine, glass beads, Bunsen burner, inoculation loop, microscope, yeast extract milk agar, VRBA (violet red bile agar) etc.

METHODS

Actually the whole experimental process was carried out in three laboratory sessions in which we were divided into three groups namely:

Process team (does all the process related work)

Microbiology team (does all the microbiology related analysis various samples)

Analytical team (does analysis of various components from samples)

We were divided into groups because it helps in a smooth and organised way of the process and thus promoting team work in the students. I was happened to be a member in the microbiology team and hence we dealt with only the microbiological analysis of milk, cheese and curd.

OVERVIEW OF PROCESS

The raw milk was inoculated with lactic starter culture (streptococcus cremoris/lactobacillus lactis), Annatto (red colouring compound) and also Rennet (obtained from calf intestine) for formation of coagulum. This coagulum was cut into pieces and then cooked for certain time to get curd and whey. Then whey was separated from the curd. And curd was processed into cheese through salting, forming & maturing. The separated whey was subjected to ultra filtration, which resulted in two components:

Whey permeate

Whey retentate

Whey retentate can be further processed to obtain whey proteins which are used extensively in food stuff. Further some yeast inoculum was added to the whey permeate to initiate the process of fermentation. This was done with two samples A and B (in which extra lactose was added externally). The fermentation was carried out for 5 days and for every one day the samples were collected and kept aside for the analysis of lactose and protein. After the fermentation, both samples were subjected to centrifugation. This results in ethanol (whey wine) and also yeast biomass (pellet). The yeast biomass was further processed to obtain yeast extract, a food additive with wide range of applications in food industry. The ethanol was further subjected to acetification, which results in formation of acetic acid using Acetic acid bacteria (Acetobacter aceti). This Acetic acid was clarified using charcoal column to get vinegar. (Walker, G. and White, N. 2009 pers. Comm., 16 November)

MICROBIOLOGICAL ANALYSIS OF MILK

ENUMERATION OF VIABLE BACTERIA (GENERAL VIABLE COUNT)

For this method, the suggested medium was Yeast extract milk agar (a medium which selectively supports the growth of microorganisms associated with milk & its products).

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

1 in 10 dilution = 9 ml water + 1 ml milk

1 in 100 dilution = 9 ml water + 1 ml of 1 in 10 dilution

1 in 1000 dilution = 9 ml water + 1 ml of 1 in 100 dilution

During these dilutions at every step each test tube was mixed to make the solution uniform. According to the Scottish regulations, only 1 in 100 and 1 in 1000 dilution samples were tested. 1 ml of 1 in 100 and 1 ml of 1 in 1000 dilution samples were placed in two separate Petri dishes and afterwards 10 ml of malted agar was poured into the Petri dish. After the solidification of medium, the plates were incubated in inverted position for 72 hours at 30 degrees centigrade. After the incubation, the number of colonies in each plate were counted using colony counter and it was then multiplied by the dilution, and the resultant is called “number of viable bacterial per ml”.

TOTALCOLIFORM COUNT

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

The serial dilutions were done as for the first method. Then using overlay method, first pour 1 ml of 100 and 1ml of 1 in 1000 dilutions in Petri plates and then 8 ml of fresh molten VRBA was poured. It was allowed to set and finally it was overlaid with 3-4 ml of fresh molten VRBA.

Overlay method ensures anaerobic conditions which suppress growth of non fermentative gram negative bacteria. It also encourages the lactose fermentation which ultimately favours formation of visible purple colonies.

The Petri plates were incubated at 37 degrees centigrade for 72 hours. After the incubation, the Petri plates were used to count the coliforms which were present in the visible purple colour colonies. Coliform count was done as colony forming units (CFU) per ml milk.

MICROBIOLOGICAL ANALYSIS OF CHEESE & CURD

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 process was used for the microbiological analysis purpose. The analysis for both of them was done same as that of milk, but with some minor changes. Here instead of 1 ml of milk, 1 gram of curd as well as cheese were used for the serial dilution purpose. Also, the 9 ml blank solution was added with some glass beads. Also the dilution for cheese and curd were 1 in 100, 1 in 1000, and 1 in 10000. And similar to the milk, general viable counts and total coliform counts were performed.

RESULTS

Table 1: Calculations for the viable count of microorganism in tested samples.

Tested Samples

Dilutions

10-2 10-3 10-4

Average of colony count (cfu/ml)

Milk

1.0 x 103cfu/ml

7.0 x 103cfu/ml

n/a

8.0 x 103

Curd

1.52 x 104cfu/ml

1.12 x 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 viable colony count for milk was calculated as 8.0 x 103 CFU/ml

Average viable colony count for curd was calculated as 9.0 x 105 CFU/ml

Average viable colony count for old cheese was calculated as 3.54 x 105 CFU/ml

Table 2: Calculations for the coli form count in tested samples.

Tested Samples

Dilutions

10-2 10-3 10-4

Average of colony count (cfu/ml)

Milk

5.0 x 103cfu/ml

2.4 x 104cfu/ml

n/a

2.9 x 104

Curd

1.56 x 104 cfu/ml

9.5 x 104cfu/ml

6.3 x 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 form count for milk was calculated as 2.9 x 104 CFU/ml

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

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

CFU (colony forming units)

It is one of the unit of measurements used in microbiology which indicates the number of microorganisms present in 1 ml of the sample.

Bacterial contamination was also reported in 2 of the milk sample plates (1 in 100 dilution & 1 in 1,000 dilution).

Contaminants in milk sample plate of 1 in 100 dilution: one out of two major contaminants had characteristics like gram positive nature and cells were present in elongated. The other contaminants which were present on the peripheral region were gram positive, rod shaped and uniform in morphology. No elongated cells were present with respect to the later contaminants.

Contaminants in milk sample plate of 1 in 1000 dilution: the contaminant showed the gram positive nature and microscopic examination revealed that the cells were in the form of rod shaped and some of the cells still attached to each other.

RESULTS FROM THE ANALYTICAL TEAM

Figure 1: estimation of lactose concentration using DNS methods

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

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

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

Figure 5: Analysis of ethanol and total acid in sample A

Figure 6: Analysis of ethanol and total acid in sample B

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

GAS CHROMATOGRAPHY RESULTS FOR VINEGAR SAMPLES A & B

SAMPLE B SAMPLE A

Vial # 1 = 6.471% ethanol (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 (not

detected )

DISCUSSION

According to the Scottish regulations there must be less than15000 bacteria per ml in the premium milk, and with the bacterial count obtained in our analysis were around 8000 bacteria per ml of milk. This clearly shows that the milk is free from bacterial contamination, but there is another condition to be satisfied i.e. there must be no coli form bacteria in 1 in 100 and 1 in 1000 dilution milk samples. But unfortunately might be due to poor handling conditions, we observed contamination in both the samples. After realizing that there was contamination in the samples, the contaminating bacteria was analysed under the microscope using the gram’s staining. We discovered that the organisms that contaminated the samples were both gram positive, rod shaped organisms. One of them was suspected to be a species of Bacillus coagulans, a lactic acid forming bacteria and also acts a probiotic. And the other one was like mixed proteolytic bacteria with an antibiotic kind of activity surrounding it in the Petri dish. One more observation that was made is the number of bacterial count in curd was higher than that of milk and also old cheese indicating that more number of organisms are survived in curd, may be most of them beneficial to humans!!

CONCLUSION

The experiment proved that milk can be successfully converted into cheese, vinegar and yeast extract. Inexpensive whey was converted into expensive vinegar and ethanol and it is very novel and industrially viable process. Also attention could further be paid towards improvement of more efficient yeast strains which can use low lactose levels to give maximum output. Dividing the students into groups for carrying out various sections of the experiment was very good and thus encourages team work in them. Overall the whole process carried out was good but if the students were taken to some industry, they can get an actual picture of how the process is done in industrial scale and helps in better understanding of this lab and adds meaningfulness to it.

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