*3.2.2. Long and thin–rods with blunted ends cell morphology*

290 Lactic Acid Bacteria – R & D for Food, Health and Livestock Purposes

taken at 1000 magnifications.

RB, taken at 1000 magnifications.

Phase Contrast Micrographs of *Bifidobacterium* reference strains: **Fig. 4.1**, *B. adolescentis* on modified MRS; **Fig. 4.2**, *B. adolescentis* on RB; **Fig. 4.3**, *B. infantis* on modified MRS and **Fig. 4.4**, *B. infantis* on RB,

Phase Contrast Micrographs of the isolate strains: **Fig. 4.5**, *B. longum* GB-03 on modified MRS; **Fig. 4.6**, *B. longum* GB-03 on RB; **Fig. 4.7**, *B. bifidum* WN-04 on modified MRS and **Fig. 4.8**, *B. bifidum* WN-04 on This was the most common type of morphology encountered among the *Bifidobacterium* isolates of *B. longum* GB-03 and *B. bifidum* WN-04 on the unmodified MRS agar (Figures 4.5, 4.7, 4.9 & 4.10). Since only the general cell structure was used to differentiate this species from the other bacteria, PCM proved sufficient for this purpose. Variations of morphology within these small groups were visible under PCM as indicated by the following examples. The isolate of *B. longum* GB-03 in Figure 4.12 exemplified the diversity of rods and coccus cells morphology including bifid structures also; with the absence of any coccus build cells when grown on RB agar in Figure 4.6. By comparison with the reference strains, the cells morphology of *B. longum* GB-03 isolate is more peculiar to that displayed by *B. infantis* (Figure 4.4) and the isolate of *B. bifidum* WN-04 (Figures 4.7 & 4.8). All the *Bifidobacterium* isolates displayed long and short club-shaped rods, most of which were long and thin with blunted ends and of conventional "V" and/or "Y"-shaped cells.

### **3.3. Confirmation of identity of** *Bifidobacterium* **strains**

### *3.3.1. Fructose-6-Phosphate Phosphoketolase (F6PPK) verification test*

F6PPK is certainly a key enzyme in the "bifidus pathway" and it allows the discrimination of the specific feature on expression of fructose-6 phosphate in cellular extracts that assigned the bifidobacteria to the genus level (Sgorbati, 1979).

The procedure to test for the F6PPK activity in the *Bifidobacterium* strains is still practised as described by Scardovi (1986). In brief, cells harvested from 10 ml RB or MRS broth are washed twice with 50 mM phosphate buffer (pH 6.5). The cells are disrupted by sonication in the cold, and 0.25 ml of each of NaF and Na iodoacetate solution and fructose-6 phosphate (Na Salt: 70% purity) are added to the sonicate. The reaction is stopped by the addition of 1.5 ml of hydroxylamine HCl, and 1 ml each of trichloroacetic acid and 4 M HCl. Finally, 1.0 ml of a colour-developing agent (FeCl3.6H2O 5% (w/v) in 0.1 M HCl) is added. A tube without fructose-6-phosphate serves as a blank, to facilitate the visual comparison. The formation of acetyl phosphate from fructose-6-phosphate, shown by the reddish violet colour formed by the ferric chelate of its hydroxamate is an indicator for F6PPK. This is the distinctive and key enzyme of the "bifid shunt" that characterizes the genus. There are three subtypes of F6PPK in bifidobacteria as shown in Figure 5.

*Bifidobacterium* in Human GI Tract:

Screening, Isolation, Survival and Growth Kinetics in Simulated Gastrointestinal Conditions 293

*B. bifidum***(1)** *B. longum***(1)** *B. infantis***(1)** *B. adolescentis***(2)**

Many of the *Bifidobacterium* species groupings are heterogeneous and the entire genera have been re-examined using DNA-DNA hybridization. A point is made here that, instant phenotypic characterization of most bacteria within their respective genera relies on biochemical tests such as the proportion of acetic and lactic acid relative to the end product of metabolism; the ratio of acetic and lactic acid produced; some key carbohydrate fermentations; colonies and phenotypic morphologies; and the presence of fructose-6-

phosphate phosphoketolase (F6PPK), a key enzyme in the bifidus pathway.

Spore forming – – – – Motility – – – – Gram reaction + + + +

pleiomorphic + + + + Anaerobic growth + + + + Aerobic growth – – – – Gas from lactose – – – – Catalase – – – – F6PPK + + + +

production (ratio 3:2) + + + +

Cellobiose – + – + Fructose + + + + Fructooligosacharides – + + + Galactose + + + + Glucose + + + + Isomaltose – + + + Lactose + + + + Maltitol(3) – – – – Mannose – – – – Melezitose – – – – Raffinose + + + + Stachyose + + + + Trehalose – – – – Xylose – + – + Legends on Table 1: (1) Obtained from American Type Culture Collection, Rockville, USA. (2) Obtained from China General Microorganisms Culture Collection Center, Beijing, China. (3) Maltitol is still widely used as a non-cariogenic sweetener and sugar substitute but is as yet not used as a possible prebiotic. + positive results or fermentation; −

negative results or no fermentation observed. F6PPK (fructose-6-phosphate phosphoketolase).

**Table 1.** Phenotypic characteristics of some of the pH- and bile salts-resistant bifidobacteria tested.

**Characteristics** *Bifidobacterium* **Strains**

Morphology: rods,

Acetic and lactic

Carbohydrates Fermentation Test

### *3.3.2. Determination of acetic and lactic acids*

One possible method of validating the presence of acetic and lactic acids in the fermented milk by bifidobacteria can be assayed by using High Performance Liquid Chromatography (HPLC). Samples for this analysis are prepared by using a modified method described by Dubey & Mistry, (1996).

**Figure 5.** Fermentation of hexose for carbohydrate metabolism (the "bifid shunt"), based on Schlegel (1993), where PK, phosphoketolase; TA, transaldolase; TK, transketolase, Ac~P, acetyl phosphate; GAP, glyceraldehydes-3-phosphate.

The strains were maintained anaerobically by propagation in MRS broth (peptone: 10 g/l; meat extract: 8 g/l; yeast extract 5 g/l; D(+)glucose: 20 g/l; di-potassium hydrogen phosphate: 2 g/l; di-ammonium hydrogen citrate: 2 g/l; Tween-80: 1 ml/l; sodium acetate: 5 g/l; magnesium sulfate: 0.2 g/l; manganese sulfate: 0.04 g/l, supplemented with 0.05% (w/v) cysteine-hydrochloride).

The production of acetic and lactic acids, spore formation, aerobic and anaerobic growth, gram reactions, motility, gas production from lactose and carbohydrates fermentation tests are some of the confirmation tests that proves highly diagnostic personality characteristics of different *Bifidobacterium* spp as summarized in Table 1. Furthermore, the taxonomy of bifidobacteria has changed ever since they were first isolated. They had been assigned to the genera *Bacillus*, *Bacteroides*, *Nocardia*, *Lactobacillus* and *Corynebacterium* among others, before being recognized as a separate genus in 1974.

Many of the *Bifidobacterium* species groupings are heterogeneous and the entire genera have been re-examined using DNA-DNA hybridization. A point is made here that, instant phenotypic characterization of most bacteria within their respective genera relies on biochemical tests such as the proportion of acetic and lactic acid relative to the end product of metabolism; the ratio of acetic and lactic acid produced; some key carbohydrate fermentations; colonies and phenotypic morphologies; and the presence of fructose-6 phosphate phosphoketolase (F6PPK), a key enzyme in the bifidus pathway.

292 Lactic Acid Bacteria – R & D for Food, Health and Livestock Purposes

subtypes of F6PPK in bifidobacteria as shown in Figure 5.

*3.3.2. Determination of acetic and lactic acids* 

Dubey & Mistry, (1996).

glyceraldehydes-3-phosphate.

cysteine-hydrochloride).

being recognized as a separate genus in 1974.

The procedure to test for the F6PPK activity in the *Bifidobacterium* strains is still practised as described by Scardovi (1986). In brief, cells harvested from 10 ml RB or MRS broth are washed twice with 50 mM phosphate buffer (pH 6.5). The cells are disrupted by sonication in the cold, and 0.25 ml of each of NaF and Na iodoacetate solution and fructose-6 phosphate (Na Salt: 70% purity) are added to the sonicate. The reaction is stopped by the addition of 1.5 ml of hydroxylamine HCl, and 1 ml each of trichloroacetic acid and 4 M HCl. Finally, 1.0 ml of a colour-developing agent (FeCl3.6H2O 5% (w/v) in 0.1 M HCl) is added. A tube without fructose-6-phosphate serves as a blank, to facilitate the visual comparison. The formation of acetyl phosphate from fructose-6-phosphate, shown by the reddish violet colour formed by the ferric chelate of its hydroxamate is an indicator for F6PPK. This is the distinctive and key enzyme of the "bifid shunt" that characterizes the genus. There are three

One possible method of validating the presence of acetic and lactic acids in the fermented milk by bifidobacteria can be assayed by using High Performance Liquid Chromatography (HPLC). Samples for this analysis are prepared by using a modified method described by

**Figure 5.** Fermentation of hexose for carbohydrate metabolism (the "bifid shunt"), based on Schlegel (1993), where PK, phosphoketolase; TA, transaldolase; TK, transketolase, Ac~P, acetyl phosphate; GAP,

The strains were maintained anaerobically by propagation in MRS broth (peptone: 10 g/l; meat extract: 8 g/l; yeast extract 5 g/l; D(+)glucose: 20 g/l; di-potassium hydrogen phosphate: 2 g/l; di-ammonium hydrogen citrate: 2 g/l; Tween-80: 1 ml/l; sodium acetate: 5 g/l; magnesium sulfate: 0.2 g/l; manganese sulfate: 0.04 g/l, supplemented with 0.05% (w/v)

The production of acetic and lactic acids, spore formation, aerobic and anaerobic growth, gram reactions, motility, gas production from lactose and carbohydrates fermentation tests are some of the confirmation tests that proves highly diagnostic personality characteristics of different *Bifidobacterium* spp as summarized in Table 1. Furthermore, the taxonomy of bifidobacteria has changed ever since they were first isolated. They had been assigned to the genera *Bacillus*, *Bacteroides*, *Nocardia*, *Lactobacillus* and *Corynebacterium* among others, before


Legends on Table 1: (1) Obtained from American Type Culture Collection, Rockville, USA. (2) Obtained from China General Microorganisms Culture Collection Center, Beijing, China. (3) Maltitol is still widely used as a non-cariogenic sweetener and sugar substitute but is as yet not used as a possible prebiotic. + positive results or fermentation; − negative results or no fermentation observed. F6PPK (fructose-6-phosphate phosphoketolase).

**Table 1.** Phenotypic characteristics of some of the pH- and bile salts-resistant bifidobacteria tested.
