Starter culture and their purity determination

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2. Lactic Acid Bacteria
• All dairy fermentations use lactic acid
bacteria(LAB) for acidification and flavor
production.
• Common characteristics of LAB
– gram-positive
– non–spore forming
– non pigmented
– Catalase negative
– aerotolerant
– ferment lactose with lactic acid as a major end
product.
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3. Quality Control Tests
• The ability of a starter culture to perform its
function during the manufacture of fermented
dairy products depends on its purity and activity.
• The purity of starter culture can be ascertained by
microscopic examination using staining methods
• The term activity refers to the ability of starter
cultures to produce desirable changes in
fermented dairy products.
• Usually activity measurements are confined to the
ability of starter cultures to acidify milk.
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5. Microscopic Examination
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Preparation of Smear

7. Simple Staining
• Place the fixed smears on a
staining loop or rack over a sink
or other suitable receptacle.
• Stain one slide with alkaline
methylene blue for 1 minute; one
slide with carbolfuchsin for 5 to
10 seconds; and one slide with
crystal violet for 20 to 30 seconds.
• Wash stain off slide with water
for a few seconds.
• Blot slide dry with bibulous paper.
• Be careful not to rub the smear
when drying the slide because
this will remove the stained
bacteria.
• Examine under the oil immersion
lens.
• You may observe cells in one of
the shapes and arrangements
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12. Lactobacillus casei
Lactobacillus bulgaricus
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13. Special Staining
• Bacterial Spore Bacterial Capsule Staining
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i)Tube method:
1.Add 1.0 ml of hydrogen peroxide (3 %) to about 5 ml
of starter culture in test tube.
2.The appearance of gas bubbles (figure indicates a
positive test; the absence of gas bubbles is a
negative test.
3.As Lactic Acid Bacteria are catalase negative, a
positive reaction indicates gross contamination.
Catalase Test

15. Slide Method
i) Slide method: An alternative procedure for doing the
catalase test is to perform by slide method.
ii)Transfer two to three drops of actively growing culture
on to a clean glass slide and equal number of drops of
3% hydrogen peroxide and mix the content.
iii)Observe for effervescence which indicated a positive
catalase reaction, whereas no effervescence is taken
as negative test .
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17. Activity
Test
An activity or vitality test helps to
determine the rate of acid
development by a starter culture prior
to its use in the processing vat-for
example, a simulated cheese making
process in the laboratory.
For determining the activity of starter
cultures the following tests should be
performed in the quality control
laboratories:
1.Titratable Acidity Test
2.Resazurin Reduction Time Test
3.Voges Proskauer test
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18. Determination of Titratable Acidity
• The activity of starter is directly related to its ability to
increase titratable acidity in milk during specified interval of
time.
• However, for better reliability and reproducibility, the exact
conditions chosen for the tests should be well defined.
• In case of active fast acid producers, the T.A should not be
less than 0.8% lactic acid at 30 C within 16 h.
• Horrell Elliker Test: This test is based on using a higher
inoculums i.e. 3% in autoclaved reconstituted non fat dry milk
and determining the T.A after 3.5 h at 37 C.
• Development of 0.4 % lactic acid indicates a good activity of
cultures.
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19. Procedure
– Activate the culture in skim milk tubes for inoculation
@1% in 25ml of heat treated (90 ºC, 10min) reconstituted
NFDM and incubate at 30 or 42° C.
– Measure the change in pH using pH meter 16 h and 24h of
incubation.
– Estimate the titratable acidity by taking 10 g of curd in a
porcelain dish and titrating with 0.1 N NaOH using
phenolphthalein as indicator.
– The titratable acidity is calculated and expressed as
percent lactic acid (% LA) as follows.
Acidity (% LA) = 9 X Volume of NaOH consumed (ml) X Normality
of NaOH/ Weight of curd sample taken for titration (g)
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20. Resazurin Reduction Time Test
• Preparation of Dye Solution: Autoclave 200 ml of
distilled water in light resistant flask.
• Add slightly more than 200m ml of distilled water to the
flask.
• Promptly transfer one resazurin tablet with clean dry
forcep to the flask of hot water.
• Allow the tablet to dissolve and then let it cool down.
• With pipette, transfer 1 ml of resazurin solution and
label the tube.
• Transfer 10 ml of starter culture to the tube.
• Incubate at 37 C till complete reduction of dye to leuco
compound.
• Record the reduction time
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21. The Voges-Proskauer or Creatine test
• Creatine test is a biochemical test generally carried
out to check aroma producing culture and is based
ion the ability of such cultures to produce acetyl
methyl carbinol and diacetyl, which form pink
coloured complex with creatine.
• This test is sometimes also used to detect gas-
producing organisms in a non-gas-forming culture.
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22. Procedure
• Inoculate active cultures @1% in
2ml of citrate broth.
• To this, add 1ml of 0.5%
creatine solution and 1ml of 5%
α-naphthol freshly dissolved in
2.5 N NaOH.
• Place the tubes in dark for 10
minutes and observe for
development of pink colour.
• The appearance of pink colour is
an indication of positive test for
flavor production.
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