Lab Assignment: Fermented Milk

Introduction

For millennia fermented foods remained part of the human diet. These fermented foods

required suitable microbes which on inoculation with raw material caused desirable changes

in texture, taste and odour to form the fermented product. Among which yoghurt possesses

its own starter cultures, the pair Streptococcus thermophilus and Lactobacillus bulgaricus are

considered to be the most favourable starter cultures to produce thick and tangy yoghurt.

Here, both these bacteria had the ability to ferment lactose to lactate. Both

complimented each other growth by producing optimum condition conditions and

metabolic products, explained further in the discussion. The characteristics of the bacterial

strains used in this experiment for fermentation of milk are as follows; -

Sr. No Cultures Characteristics

1. Streptococcu  Gram-positive bacteria

s  Spherical

thermophilus  Optimum condition for growth: -

1. pH – 6.5 to 6

2. Temperature – 40 °C to 45 °C

2. Serratia  Gram-negative bacteria

plymuthica  Rod-shaped

 Can slowly ferment lactose

 Spoilage bacteria which produce

enzymes like DNase, protease and lipase

 which degrade food.

 Optimum condition for growth: -

1. pH – 7 to 9

2. Temperature – 20 °C to 37 °C

3. Lactobacillus  Gram-positive bacteria

bulgaricus  Rod-shaped

 Optimum condition for growth: -

1. pH – 5.4 to 4.6

2. Temperature – 40°C to 44 °C

Table 1: Characteristics of microbes used for milk fermentation (Wikipedia,2023)

The strains used were facultative anaerobe and except Serratia plymuthica all others were

Lactic acid bacteria. The detailed interaction of bacteria with each other is explained in the

discussion section. However, the samples containing Streptococcus thermophilus and

Lactobacillus bulgaricus should end up forming yoghurt because they not only ferment

lactose and make a thick product but also incorporate that tangy acidic flavour into it(K.

Mchiouer*, 2017)..

From (Table 1) it was presumed that cultures Sample A, D and E would give a thick set

yoghurt-like consistency while the rest of them would fail to do the following. Since the

above three samples consisted of Streptococcus thermophilus; whose conditions for optimal

growth were fulfilled. Also, Serratia plymuthica had no significant contribution in yoghurt

formation as it was not suitable for this process which was reflected in the experiment.
 Instruments such as the thermometer and pH meter were used to check the optimal

temperature of milk before inoculation and pH in yoghurt combinations. Visual and

Olfactory means came in handy to observe this sensory aspect of fermented milk samples.

One could also try to taste the yoghurt samples to give a precise opinion about their taste.

Lactobacillus acidophilus could be used with the above standard starter culture to give

probiotic properties to the yoghurt which include improved gut digestion and boosting

immunity(Katherine Marengo LDN, 2018). Some people prefer yoghurt with a thin

consistency for this Lactobacillus acidophilus, and Lactobacillus helveticus are added to the

traditional starter mix forming yoghurt which is thin and tangy. They are thin because this

strain produces less exo polysaccharide which is crucial for the thinness of yoghurt. Also,

Lactobacillus helveticus promotes improved gut health and mitigates high blood

pressure(Debra Rose Wilson, 2019).

Methods and Materials

Glassware Equipment

Plastic test tubes – x 14 Boiling water bath

Glass pipette – x 2 Microscope

Glass slides – x 4 Bunsen burner

Sterile Container (200 ml) – x 1 Incubator

For Yoghurt mixture Miscellaneous

Sterile Pasteurized Milk - 112 mL Micropipette (1000mL) – x 1

Starter cultures: (1% solution) Micropipette tips – x 14

1. Streptococcus thermophilus Nichrome loop

2. Lactobacillus bulgaricus Sterile water

3. Serratia plymuthica Gram stains

Ethanol

Droppers – x 5

Paraffin oil

Pencil

Lighter

Table .2: Materials used during the milk fermentation experiment

1. First milk was boiled at 42 °C in a Boiling water bath. It had a neutral pH of 6.5.

2. The 14 universal containers were labelled as A1, B1, C1, D1, E1, F1, G1, A2, B2, C2,

D2, E2, F2, G2.

3. 8mL of milk was added to each container aseptically.

 4. 0.3 mL of respective cultures and their combinations were added to their assigned

containers using a micropipette. The addition would be done according to the table

given below: -

A1 & A2 B1 & B2 C1 & C2 D1 & D2 E1 & E2 F1 & F2 G1 &

G2

Streptococc Serratia Lactobacill Streptococc Streptococc Lactobacill No

us plymuthi us us us us culture

thermophil ca bulgaricus thermophil thermophil bulgaricus s

us us and us and and

Lactobacillu Serratia Serratia

s bulgaricus plymuthica plymuthica

Table 3: Pattern of adding starter cultures (inoculum) in milk for overnight fermentation

5. The proper swirling of the containers followed this to ensure the proper mixing of

bacteria in milk.

6. Then these mixtures were incubated at 42 °C for 21 hours.

7. Later, these samples' odour and consistency/ texture were observed. Their pH was

measured using a pH meter.

8. Finally, Gram staining was carried out for analysis of microbiota developed in yoghurt

after fermentation
 Results

Samples pH Average pH Texture Odour

A1 4.67 4.68 Thick, gel - like Yoghurty

A2 4.70

B1 6.19 6.18 Biofilm formation, Pungent, Milk-like

B2 6.17 Thinner than milk

C1 6.01 6.02 Slight coagulation, Milk like

C2 6.03 but overall Milky

D1 4.01 4.02 Thick, gel-like Same as commercial

D2 4.03 yoghurt

E1 5.03 5.04 Thick, gel-like Yoghurty, less acidic

E2 5.05

F1 5.80 5.85 Milky Milk-like, slightly

F2 5.91 acidic, bit spoilt

G1 6.61 6.65 Milky Milky

G2 6.7

Table 4: A table to show the change in pH, texture and odour of fermented milk after

incubation with respective culture for 21 hours at 42° C

(Note – Each sample had its duplicate, but the results were discussed by taking an average of

data and by considering only one type of sample and not its duplicate)
Among these; Samples A, D, E had thick (yoghurt-like) consistency while the rest of them had

an overall milky texture (few of them had slight coagulation in them). Among these sample A

had the lowest pH while Sample G had greater pH than any other samples.

Discussion

In Sample A, milk was inoculated by Streptococcus thermophilus as expected it was able

to ferment lactose sugar into lactic acid, which caused a drop in pH of fermented milk

(yoghurt) from 6.65 to 4.68 (Table 1). Casein micelle's composition consisted presence of

calcium phosphate which maintained the rheological properties of milk. The phosphate

present in it made casein micelle a negatively charged molecule which existed in milk in its

soluble form(Walstra, 1999). When lactic acid was produced by this microbe the casein

micelle became insoluble, because the lactic acid dissociated in protons and reacted with

negatively charged casein micelle, which ultimately caused the casein micelle to reach its

isoelectric point and turn them insoluble in milk. The absence of calcium phosphate caused

the casein micelle to come together as there was no negative charge to maintain that

repulsion within casein micelles (Note – Similar reaction happens in Sample D and E with

slight variation).

Fig 1: Represent the changes happening in

the structure of casein micelle as the pH

changes from neutral to acidic. (Kaya-

Celiker and Mallikarjunan, 2012) (highs, 2013)

Even this was a reason which caused thickening and gave yoghurt its gel-like texture. (De

Vuyst et al., 2003) depicted that Streptococcus thermophilus (as well as Lactobacillus
bulgaricus) a Lactic Acid Bacteria (LAB) had a unique characteristic where it produced

exopolysaccharides (EPS) which contributed to the elevated viscosity of yoghurt, however

the degree of EPS production was more in streptococcus which is why sample C and F had

thin consistency in absence of it. The milk provided the optimum conditions for the

proliferation of this microorganism. Compared to the normal yoghurt this sample was

slightly less acidic but had that typical set yoghurt-like consistency.

Serratia plymuthica was unable to cause any significant textural or chemical change in the

milk sample (concerning yoghurt production) since it was a spoilage bacteria compared to

LAB it had a low ability to ferment lactose into lactate. Due to this, the pH change in sample

B was around 0.07 which was very low as compared to samples A and B (6.25 to 6.18). It

smelt a bit pungent since this bacterium was capable of breaking milk fats and protein by

producing lipolytic and proteolytic enzymes. Interestingly, this sample had a thin biofilm

formation above the milk sample which was a distinct feature of this bacteria {Cleto, 2008

#100}. Although after 21 hours of incubation sample B had a milk-like consistency.

Despite being a Lactose fermenting bacterium, Lactobacillus bulgaricus was unable to

cause a significant decrease in the pH of milk which ultimately resulted in no yoghurt

formation. This is because this bacterium needed an acidic pH to multiply rapidly also the

absence of some other metabolites (like formate) resulted in its limited growth,

comparatively, it produced low exopolysaccharides. Hence, this bacterium is usually paired

with Streptococcus thermophilus which creates the optimum surrounding for its

multiplication. Although it caused some coagulation of casein micelle it was inadequate to

produce thick yoghurt. The pH dropped from 6.5 to 6.02 which is not sufficient to produce

thick yoghurt.
 Sample D was a commercially used starter culture which consisted of a combination of

two Gram-positive bacteria namely Streptococcus thermophilus and Lactobacillus bulgaricus.

They shared a symbiotic relationship where both produced certain compounds which

stimulated the growth of other bacteria. Streptococcus thermophilus produced formate

which stimulates the growth of Lactobacillus bulgaricus for the efficient production of lactic

acid (K. Mchiouer*, 2017). Lactobacillus bulgaricus formed free amino acids and peptides

(from milk proteins) which are used by Streptococcus thermophilus for efficient

multiplication. This duo brought the pH of the fermented milk (yoghurt) to 4.02, which gave

it that typical Yoghurty smell and thick consistency. That is why this starter culture is

extensively used by food manufacturers to produce milk-based fermented products like

yoghurt.

Optimal conditions were present for Streptococcus thermophilus for multiplication while

Serratia plymuthica showed gradual growth in milk since the pH was not optimum for its

proliferation (Table 1). Streptococcus thermophilus consistently produced lactate which

caused a pH drop and led to coagulation of casein micelle and formed thick yoghurt.

Serratia plymuthica did not cause any effect on its counterpart because Streptococcus

immediately started producing lactate and made the milk acidic which was unfavourable for

optimal growth of Serratia. So, Streptococcus was unaffected by its bacteriostatic effect

Sample D had a pH of 5.03 which was more than sample D, which indicated the fact that

there was low lactate production in Sample E as compared to Sample D (Table 4). When

observed under the microscope pink rod-shaped Serratia colonies were quite low as

Streptococcus were quite more because proteases produced by Serratia plymuthica had a

proteolytic effect on milk proteins, and the peptides or amino acids produced from it were
used by Streptococcus for its proliferation. The odour from this sample was not acidic as

compared to Sample D and A, it smelt more like milk with a faint hint of sourness (Table 4).

In Sample F, both Lactobacillus bulgaricus and Serratia plymuthica were unsuccessful in

causing any significant change in the pH of the milk. Hence, there was no coagulation of milk

and yoghurt was not produced. As (Horiuchi and Sasaki, 2012) explained Lactobacillus find it

challenging to grow in environments with high oxygen and the optimum pH was not met

(Table 1), it produced some amount of lactic acid which was simply insufficient for causing

any textural change in milk. However, compared to samples B and C, this particular mixture

of microorganisms produced more lactic acid since Serratia was marginally competent to

ferment lactose (even though it was inferior to LAB). Overall Sample F had a milky texture

with a slightly rancid odour. Due to the degradation of milk proteins.

Sample G wasn’t exposed to any microbial starter culture, rather it was used as a negative

control. So naturally there was no microbial growth found in this sample when observed

under the microscope. It retained its milky consistency after incubation and smelt like

normal milk. There was no change in its pH which was neutral, around 6.5 (Table 4).

Conclusion

Among all the mixtures, Sample D was equivalent to commercially available yoghurt since

it had a consistency, taste and aroma similar to it. Even though sample E has the texture but

lacked that acidic flavour of yoghurt. Sample B consisted of Serratia plymuthica which was

inappropriate for yoghurt formation as it spoiled it and degraded its texture and odour. From

this, it was concluded that only one type of lactic acid bacteria wasn’t enough to bring that

yoghurty property, rather using multiple strains is crucial for the production of this

fermented food.
References

Pothuraju, R., Yenuganti, V. R., Hussain, S. A. and Sharma, M. (2018) 'Chapter 29 -

Fermented Milk in Protection Against Inflammatory Mechanisms in Obesity', in Chatterjee,
S., Jungraithmayr, W. and Bagchi, D. (eds.) Immunity and Inflammation in Health and
Disease: Academic Press, pp. 389-401.

K. Mchiouer*, S. B., M. Meziane (2017) 'Microbial interactions between Lactobacillus

Bulgaricus and
Streptococcus Thermophilus in milk', Journal of Materials and
Environmental Sciences
ISSN : 2028-2508, 8, pp. 1460-1466.

Harnett, J., Davey, G., Patrick, A., Caddick, C. and Pearce, L. (2011) 'Lactic Acid Bacteria |
Streptococcus thermophilus', in Fuquay, J.W. (ed.) Encyclopedia of Dairy Sciences (Second
Edition). San Diego: Academic Press, pp. 143-148.

Cooney, S., O'Brien, S., Iversen, C. and Fanning, S. (2014) 'Bacteria: Other Pathogenic
Enterobacteriaceae – Enterobacter and Other Genera', in Motarjemi, Y. (ed.) Encyclopedia
of Food Safety. Waltham: Academic Press, pp. 433-441.

Wikipedia contributors. (2023, December 5). Serratia. In Wikipedia, The Free Encyclopedia.
Retrieved 21:52, December 5, 2023, from https://en.wikipedia.org/w/index.php?
title=Serratia&oldid=1188430708
Wikipedia contributors. (2023, August 26). Lactobacillus delbrueckii subsp. bulgaricus.
In Wikipedia, The Free Encyclopedia. Retrieved 21:44, December 5, 2023,
from https://en.wikipedia.org/w/index.php?
title=Lactobacillus_delbrueckii_subsp._bulgaricus&oldid=1172390849
Katherine Marengo LDN, R. D. (2018) Which yogurts contain Lactobacillus acidophilus?:
Medical News Today. Available at:
https://www.medicalnewstoday.com/articles/323364#yogurt-brands-that-contain-l-
acidophilus.

Debra Rose Wilson, P. D., MSN, R.N., IBCLC, AHN-BC, CHT (2019) 16 Benefits of Lactobacillus
Helveticus: healthline. Available at: https://www.healthline.com/health/lactobacillus-
helveticus.

Walstra, P. (1999) Dairy technology : principles of milk properties and processes. New York:
Marcel Dekker, p. 135,137,139.

Tamime, A. Y. and Robinson, R. K. (2007) Tamime and Robinson's yoghurt : science and
technology. 3rd edn., Cambridge
Boca Raton Fl.: CRC Press
bWoodhead Publishing, p. 94 and 95.

De Vuyst, L., Zamfir, M., Mozzi, F., Adriany, T., Marshall, V., Degeest, B. and Vaningelgem, F.
(2003) 'Exopolysaccharide-producing Streptococcus thermophilus strains as functional
starter cultures in the production of fermented milks', International Dairy Journal, 13(8), pp.
707-717.

Nishimura, J. (2014) 'Exopolysaccharides Produced from <i>Lactobacillus

delbrueckii</i> subsp. <i>bulgaricus</i&gt', Advances in Microbiology,
Vol.04No.14, pp. 7.

Cleto, S.Simões, M.Matos, S. V. and J, M. (2008) 'Serratia plymuthica dairy industry isolates
and their antimicrobial metabolites impact on pathogens', INTERNATIONAL SYMPOSIUM ON
MICROBIAL ECOLOGY, 12, Cairns, Australia, 2008.

Kaya-Celiker, H. and Mallikarjunan, P. (2012) 'Better Nutrients and Therapeutics Delivery in

Food Through Nanotechnology', Food Engineering Reviews, 4.

highs, F. (2013) 'Kitchen Chemistry – Say Cheese!'. Available at:

https://www.foodhighs.com/2016/07/11/kitchen-chemistry-say-cheese/.

Petersen, L. M. and Tisa, L. S. (2014) 'Molecular characterization of protease activity in

Serratia sp. strain SCBI and its importance in cytotoxicity and virulence', J Bacteriol, 196(22),
pp. 3923-36.

Horiuchi, H. and Sasaki, Y. (2012) 'Short communication: Effect of oxygen on symbiosis

between Lactobacillus bulgaricus and Streptococcus thermophilus', Journal of Dairy Science,
95(6), pp. 2904-2909.