Food Biochemistry and Food Processing (2 edition)

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676 Part 6: Health/Functional Foods

(e.g., isoflavones), and enzymes are but a few of the many com-

pounds have been suggested as being linked to functionality of

probiotics. In this chapter, the potential contribution of enzymes

produced by probiotic bacteria to their functionality will be ex-

amined. The critical contribution of enzymes will be addressed

from both a technological perspective (ability to grow and re-

main viable in a food product) and a health perspective (survival

to GI environments, effects on human functions).

BIOCHEMISTRY AND GROWTH OF

PROBIOTIC CULTURES IN FOODS

Several different metabolic pathways explain the biosynthetic

functions of microorganisms that could maintain their life. Each

metabolic pathway consists of many reactions that are regulated

by different enzyme systems, and so the level of enzymes and

their activity sustain and control the functions of the microbial

cell (Stanier et al. 1987). Breakdown of nutrients (carbohydrates,

proteins, lipids, and other minor constituents) present in the

growth medium results in the production of smaller molecules

that are subsequently consumed for buildup and division of the

microbial cells as well as energy requirements for survival.

In lactic acid bacteria (LAB) (i.e., the lactococci, leuconostoc,

lactobacilli, streptococci, and bifidobacteria), energy is mainly

supplied by the fermentation of carbohydrates (Lawrence et al.

1976). Hundreds of enzymes are required to sustain life in mi-

croorganisms. However, some are recognized as being more

critical in affecting growth rates and biomass levels on food

substrates. In general, the two most important biochemical re-

actions are carbohydrate assimilation and protein metabolism.

This justifies the focus that will be made of these particular

enzymatic activities.

Milk

In milk, “lactase” and “protease” activities of yogurt and pro-

biotic cultures are critical to growth and acidification rates

(Tamime and Robinson 1999).

Lactose is the main carbohydrate in milk and it can be

metabolized either through the homo- or heterofermentative

metabolic pathways.Streptococcus thermophilus, Lactobacil-

lus delbrueckii subsp. bulgaricus,andLactobacillus acidophilus

ferment lactose homofermentatively, lactic acid being the main

metabolite.Bifidobacteriumspp., on the other hand, ferments

the same sugar heterofermentatively, producing lactic and acetic

acid typically at a 2:3 ratio. Lactose must enter the cytoplasm to

be catabolized. A specific system is involved in lactose transport

in the lactococci and certain strains ofL. acidophilusand sugar is

phosphorylated by phosphoenolpyruvate (PEP) during translo-

cation by the PEP dependent phosphotransferase system (PTS)

(Kanatani and Oshimura 1994, Marshall and Tamime 1997).

This mechanism is known as PEP:PTS.β-Phosphogalactosidase

hydrolyses lactose-6-phosphate to its monosaccharide compo-

nents. The galactose and glucose thus released are then catab-

olized via Tagatose and Emden–Meyerhof–Parnas (EMP) path-

ways, respectively (Monnet et al. 1996, Marshall and Tamime

1997). Some organisms includingBifidobacteriumspp. have an

alternative system for lactose transport into the cells that in-

volves cytoplasmic proteins (permeases). This is to translocate

lactose without chemical modification and the mechanism could

be similar to the lactose permease system inEscherichia coli.

After lactose enters the cell, it is hydrolyzed byβ-galactosidase

(β-gal) to nonphosphorylated glucose and galactose. In many

yogurt cultures, glucose is catabolized to pyruvate and the galac-

tose is secreted from the cell. Less is known on the lactases of

probiotic bacteria. Some bifidobacteria seem to have two types

ofβ-gal and their level seem critical to rapid growth in milk.

Indeed, Desjardins et al. (1991) showed that the growth perfor-

mance of bifidobacteria in milk is linked to theirβ-gal levels.

L. acidophilusorBifidobacteriumsp. generally grows faster on

synthetic media than on milk (Misra and Kuila 1991, Gaudreau

et al. 2005). Therefore, it is not surprising that the addition of

β-gal to milk enhances the growth rate ofL. acidophilus(Khattab

et al. 1986). In an interesting variation of this concept, ruptured

cells of a yogurt culture were added to milk in order to promote

the development ofL. acidophilusandBifidobacterium longum

in the fermented product (Shah et al. 1997). Presumably, the

liberation ofβ-gal from the ruptured starter cells contributed

to the stimulation. This was also noted forLactobacillus rham-

nosus(Gaudreau et al. 2005). However, the ability of a strain

to synthesize enzymes essential in the assimilation of a carbo-

hydrate provides an incomplete picture of the ability to grow

rapidly on the food matrix. The activity of the enzymes will also

influence growth rates. Thus, for a given strain, growth rates

in identical media will vary as a function of the carbohydrate

(Table 35.1). There are numerous observations of variable en-

zyme activity levels between strains (Desjardins et al. 1991,

Donkor et al. 2007).

The protein fraction in milk is composed of casein and whey

proteins and the basic constituents of a protein molecule are

compounds from 21 amino acids (Tamime and Robinson 1999).

Since LAB cannot synthesize many amino acids (Marshall and

Law 1984), proteolytic activity is greatly involved in both nu-

trition and interactions between the two yogurt cultures as well

as with probiotic bacteria. Amino acids and peptones are good

supplements for bifidobacteria (Klaver et al. 1993). This would

suggest that high proteolytic activities would enhance growth

rates and levels. This is not always the limiting factor with

Table 35.1.Effect of the Nature of the Sugar on Growth

Rate (h−^1 ) of Two Probiotic Cultures in a Laboratory

Growth Medium (Peptones, Salts, Tween)

Carbohydrate

Lactobacillus

helveticusR0052

Bifidobacterium

longumR0175

Glucose 0.13 0.06

Stachyose 0 0.06

Lactose 0.15 0.07

Fructose 0.08 0

Sucrose 0.08 0

Raffinose 0 0.06

Source: Champagne et al. 2010.