METHODOLOGY

Milk sample Collection and Transportation

Before conducting the study, an agreement of written consent will be signed with milk sellers,
after being informed about the reason for conducting the research. The samples will be
collected in sterile containers and labelled with the date of collection. All samples will be
transported to the microbiology laboratory of the School of industrial Sciences Laboratory of
Harare Institute of Technology.

Isolation of Lactobacillus strains from sour milk

Isolation of Lactobacillus will be done according to (Abid et al., 2022). MRS (De Man,
Rogosa Sharpe Agar) medium will be used for the isolation and purification of bacteria from
the milk samples. The milk samples will be spread on MRS plates by the serial dilution
method using sterile distilled water. Serial dilutions will be made up to 5 folds. 0.1ml of each
serially diluted sample will be poured into already prepared MRS medium containing
bromocresol purple and incubated at 37 °C for 48 h using an incubator.

Purification of colonies

The purification of the colonies will be carried out by using the steak plate method according
to (Goa et al., 2022). After successful growth of Lactobacillus on MRS agar, morphologically
varying/distinct colonies will be further subcultivated by streaking on new MRS agar plates
using sterile-inoculating needles and incubated at 37°C for 24-48 hours inside the incubator.
Morphological characterization, including colour, shape, margin, elevation, texture, and size,
will be determined by following Bergey’s manual. Different biochemical tests will be
performed, such as Gram staining, evaluation of catalase and fermentation for the
identification of Lactobacillus isolates. The results will be interpreted according to “Bergey’s
Manual of Determinative Bacteriology”. Single colony will be stored in MRS agar slant for
further study.

Identification of Lactobacillus from sour milk

Lactobacilli isolated from sour milk will be identified based on morphological, physiological,
and biochemical tests.

Morphological Examination

Gram staining:
Gram staining test will be performed for all isolated strains according to the standard
procedure (Mannan et al., 2017). A smear of single colony will be prepared on a clean glass
slide and the smear is allowed to air-dry and then heat fixed. The heat fixed smear will be
flooded with crystal violet solution and after one minute, it then washed with water and
flooded with mordant Gram’s iodine. The smear will be decolorized with 95% ethyl alcohol
and rinsed with water. Finally, safranin will be used as counter stains for 60-80 sec and
washed with water, and examined under oil immersion (100X).

Biochemical Identification

Catalase Test

Catalase enzymes break down hydrogen peroxide into oxygen (which is seen/visualized as
the formation of bubbles) and water molecules (2H 2O2⟶2H2O + O2). The catalase test will
be conducted by adding a drop of 3% solution of hydrogen peroxide to a glass slide on which
a colony of bacteria will be applied of a 24 hour-old culture of each isolate (or directly on the
Petri dish). NB: catalase negative bacteria will be subjected to further examination (Goa et
al., 2022).

pH tolerance test:

MRS broth at pH 2, 3, 4, 5, 6, 7 and 8 will be prepared by adjusting with 10N HCl and 1N
NaOH. Fresh bacterial cultures will be inoculated into respective MRS broth in test tubes and
incubated at 37°C for 48 h. Only media will be used as negative control. Results will be
obtained by observing turbidity of the culture media after 24 h and 48 h and no growth will
be observed in negative control (Mannan et al., 2017).

NaCl tolerance test:

NaCl tolerance of isolated Lactobacillus will be determined by using MRS broth with 2%,
4% and 8% of NaCl concentration. Fresh culture will be inoculated and incubated at 37°C for
48 h. Only media will be used as negative control. Results will be determined by observing
the turbidity after 24 h and 48 h and no growth was observed in negative control.

Kliger’s Iron Agar (KIA) test:

All isolates will be tested for KIA test to know the mode of glucose and lactose utilization.
Fresh culture will be inoculated by stabbing the butt and streaking the slant. After incubation
at 37°C for 24 h, results will be recorded for color changes of the butt or slant, H2S or other
gas production. The results will be observed as alkaline slant and acid butt for fermentation of
glucose only, acid slant and alkaline butt for fermentation of lactose only, acid in both slant
and butt for fermentation of both lactose and glucose whereas alkaline in both slant and butt
for fermentation of neither lactose nor glucose. Production of hydrogen sulphide made
blacking of the medium and the gas production give rise to bubble formation in the tube. S.
aureus will be used as positive control (Mannan et al., 2017).

Casein digestion test:

The protease activity will be performed using MRS agar plate containing 1% skim milk
solution. Bacterial cultures will be inoculated and incubated for 48h at 37°C. Clear zones
around the cultures indicate protease activity (Smibert and Krieg, 1994). Pseudomonas spp.
and Klebsiella spp. will be used as positive and negative control, respectively.

Production of scfa using fermentation and separation

Lactobacillus isolates will be inoculated in MRS broth-based media. As a control, media

Quantification

The samples will be transferred to a gas chromatography vial. Total SCFAs, acetic,
propionic, butyric, isovaleric, and valeric acids will be determined using Agilent 7820 A GC-
5977 B MSD system (Agilent Technologies), Agilent HP-5 ms capillary column
(30 m × 250 μm × 0.25 μm), and Quadripolar spectrophotometric detectors. Total SCFA,
propionate, acetate, and butyrate productions will be determined. The procedure will start at
250 °C and followed as below: Inject contents (5 μl) in a split ratio of 25:1 by the Agilent
HP-5 ms capillary column and covered with a film of 0.15 μm composed of 80.2% 1-
methylnaphthalene. The mobile phase is comprised of Nitrogen at an initial flow rate of
1 mL/min and upholding this for 1 min, next shifting to 0.8 mL/min for 1 min, changing to
0.6 mL/min for 1 min and then returning to 1 mL/min for 9.2 min. Adjust and maintain the
temperature of the FID detector at 260 °C. Subsequently, apply the flow of the Helium and
the synthetic air to 30 mL/min and 350 mL/min, respectively. Finally, adjust the temperature
program of the oven to 100 °C, provide for 7 min and rise to 200 °C at a rate of 25 °C/min
and continue for 5 min ( Pirnia et al., 2022). The SCFA concentrations were expressed as
micromoles per millilitre.