Starter Cultures and Fermented Milk Products

Module 12. Yoghurt

Lesson 17

DEFINITION, TYPES AND MICROFLORA, STARTER SYMBIOSIS, ENUMERATION OF YOGHURT STARTERS

17.1 Introduction

Yoghurt is the most popular and widespread fermented milk product in the world. The word‘yoghurt’ or ‘yogurt’ is derived from the Turkish word ‘Jugurt’. Yoghurt is a traditional food and beverage in the Balkans and the Middle East. However its popularity has now spread to Europe and many other parts of the world. The product is popular with different names in different countries. Some examples of names by which it is popular is given in Table 19.1.

Table 17.1 Yoghurt type fermented milks in various countries

17.2 Definition

The general definition of yoghurt was given by Tamime and Deeth (1980). They defined yoghurt as a product resulting from milk by fermentation with a mixed starter culture consisting only of Streptococcus thermophilus and Lactobacillus bulgaricus.

FAO/WHO(1977) gave a more legal and scientific definition of yoghurt. Accordingly "Yoghurt is a coagulated milk product obtained by lactic acid fermentation through the action of Streptococcus thermophilus and Lactobacillus bulgaricus from milk and milk products (pasteurized or concentrated) with or without the optional additions (SMP, WMP, whey powder, etc) and the final product must contain viable organism in abundance".

FAO/WHO expert committee also permitted the use of other suitable lactic acid bacteria in addition to S. thermophilus and L. bulgaricus for the manufacture of yoghurt. These organisms however, should only be used as the supplementing flora when justified.

In India, Yoghurt has been defined byPFA as under:

17.3 Yoghurt

It means a coagulated product obtained from pasteurized or boiled milk or concentrated milk, pasteurized skimmed milk and or pasteurized cream or a mixture of two or more of these products by lactic acid fermentation through the action of Lactobacillus bulgaricus and Streptococcus thermophilus. It may also contain cultures of Bifidobacterium bifidus and Lactobacillus acidophilus and other cultures or suitable lactic acid producing harmless bacteria and if added, a declaration to this effect shall be made on the label. The microorganisms in the final product must be viable and abundant. It may contain milk powder,skimmed milk powder, unfermented buttermilk, concentrated whey, whey powder,whey protein, whey protein concentrate, water soluble milk proteins, ediblecasein, and caseinates manufactured from pasteurized products. It may also contain sugar, corn syrup or glucose syrup in sweetened yoghurt and fruits in fruits yoghurt. It shall have smooth surface and thick consistency without separation of whey. It shall be free from vegetable oil/fat. animal body fat,mineral oil and any other substance foreign to milk. The product may contain food additives permitted in Appendix C. It shall conform to the microbiological requirements prescribed in Appendix D. It shall conform to the following requirements:-

Table 17.2 Requirements for yoghurt as per PFA

Provided that titratable acidity aslactic acid shall not be less than 0.85 percent and not more than 1.2 percent.The specific lactic acid producing bacterial count per gram shall not be less than 10, 00,000.

Provided further that the type of Yoghurt shall be clearly indicated on the label otherwise standards of plain Yoghurt shall apply. The Yoghurt subjected to heat treatment after fermentation at temperature not less than 65°C shall be labelled as Thermized or Heat Treated Yoghurt and shall conform to the above parameters except the minimum requirement of specific lactic acid producing count per gram;

Table 17.3 Microbiological requirements for yoghurt as per FSSA (2006)

Micro flora of yoghurt

Micro flora in yoghurt can be classified in to 3 groups.

A Essential (Starter culture)

Lactobacillus delbrueckii subsp bulgaricus

Streptococcus thermophilus

B Non-essential

L.acidophilus

Bifidobacterium bifidus

Lactococcus lactis

Lactococcus . lactis subsp diacetyl lactis

Pediococcus

These are not essential for acid production but sometimes used for other purposes. They can be added with specific label on the pack.

C Contaminants

Yeast and moulds

Coli forms

Other bacteria

These groups should not be present in the final product. They are not wanted and they may cause defects, makes product unsafe and reduce the shelf-life.

Yoghurt starters

Microflora of yoghurt culture consist of selected strains of S. thermophilus and L. bulgaricus.

Orla-Jensen (1919, 1943) made a valuable contribution to the nomenclature and identification of lactic acid bacteria. He concluded that large, rod shaped LAB present in yoghurt with high temperature range of growth belongs to genus Thermobacterium. Of these Thermobacterium bulgaricus, isolated from Bulgarian yoghurt and Thermobacteriumjugurt, isolated from English yoghurt (Lactigens) were recognized as the important components of the yoghurt microflora.

Some important properties of yoghurt cultures are given in Table 17.4

Table 17.4 Important characteristics of yoghurt starters

Qualities of an ideal yoghurt starter

1 Purity, i.e. free from contaminants

2 Vigorous growth

3 Production of the right consistency

4 Production of good flavour without off flavour

5 Stability i.e. its balance should be easily maintained

6 No tendency to induce syneresis.

7 Should not develop excessive acidityon cold storage.

8 Should have a reasonable tolerance to sugar.

9 Should be resistant to penicillin and other antibiotics.

10 Its maintenance should be easy.

11 It should be phage resistant.

Symbiosis

The observation of symbiotic relationship between S. thermophilus and L. bulgaricus in the yoghurt starter culture was first reported by Orla-Jensen.

Mechanism of Symbiosis

Lactobacillus bulgaricus has got cell wall bound proteases. It has been established that numerous aminoacids liberated from casein by proteases produced by L. bulgaricus stimulate the growth of S. thermophilus. In particular glycine and histine are produced by Lactobacillus bulgaricus.

It is demonstrated that S. thermophilus produces formic acid which stimulates the growth of L.bulgaricus. Apart from formic acid. carbondioxide, creating anaerobic conditions and pyruvic acid produced by S. thermophilus stimulategrowth of L. bulgaricus .

Effects of Symbiosis

Asa result of symbiosis of the two cultures, several benefits have been observed for the culture as well as product quality.

Fig. 17.1 Effect of symbiosis on rate of acid production in yoghurt cultures

ST = S. thermophilus, LB = L. Bulgaricus

1. Higher rate of acid production: As compared to either S. thermophilus or L. bulgaricus alone, the combination of culture, produces higher amount of acid at all times of incubation (Fig. 17.1).
   
2. Higher amount of flavour produced: In yoghurt, the major flavour producers in L. bulgaricus. In mixed culture, the acetaldehyde production of L. bulgaricus is stimulated and it produces higher amount of acetaldehyde.
3. Resistance to sugar: more than 11% sugar slows down growth of yoghurt cultures individually, but they produce acid at faster rate even with 15% sugar in combination.
   
4. Higher cell numbers: Overall, the cell count increases, but depends on the stage of incubation.

The stimulatory effect of by L.bulgaricus results in a shortened generation time of streptococci and increased numbers. As a result, the streptococci grow faster in the early part of incubation, outnumbering the lactobacilli by 3 or 4 to 1after the first hour. During this stage it drives excess O₂ and produce formate, CO₂ and lactic acid for stimulatory growth of lactobacilli. Hence in the later stage, proportion of lactobacilli increases and streptococci decreases due to inhibitory effect of lactic acid. The ratio of rod: cocci become almost 1:1 after 4 hours.

Enumeration of yoghurt bacteria

Several countries have specified standards for viable count of lactic acid bacteria in yoghurt. Hence, a suitable medium is necessary to enumerate these bacteria in the product. Differential counting can be done in two ways.

1. By counting each microbe by cultivating separately on selective media

e.g. L.bulgaricus on MRS medium or reinforced clostridia agar at pH 5.5.

S.thermophilus on LPA or M17 medium or PCMA medium at pH 7.0.

2. Use of single medium to allow the growth of both bacteria

Certain media can give differential count of both yoghurt starters, for example;

• PPY-Eriochrome agar (Trytose,proteose-peptone yeast extract, Erichrome black T-dye, agar, glucose, lactose,Tween-80). After 24h of incubation, S. thermophilus appeared as circular colonies, 1-3 mm in diameter, convex, opaque, and white-violet, often with a darker centre while L. bulgaricus appeared as transparent, diffuse colonies (4-6 mm diameter) of undefined shape,irregular edge.
• LAB medium containing lactose, typtone,meat extract, yeast autolysate, tomato juice, Tween 80, K₂HPO₄and agar using double pour plate technique. Differentiation is made by colonytype under a low power microscope. S. thermophilus gives smooth, round or lenticular colonies and L. bulgaricus gives irregular, or roughcolonies in the deapth of the medium.
• A medium in which b -glycerophosphate is incorporated. This inhibits the growth of Lactobacillus bulgaricus but not that of S. thermophilus.