4. ANÁLISIS JURÍDICO-PENAL
4.3. El artículo 145 del Código Penal
4.3.3. El artículo 145-3
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Nutrition & Food Science Vol. 36 No. 5, 2006 pp. 349-356
EEmerald Group Publishing Limited 0034-6659 DOI 10.1108/00346650610703207
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with Lactobacillus acidophilus resulted in a product, which was more efficacious than acidophilus milk in preventing the gastro-intestinal disease in infants (Nabukhotnyi et al., 1983). Reddy (1989) suggested associative application of propionic acid bacteria with Lactobacillus acidophilus, Bifidobacterium bifidum and Leuconostoc citrovorum during the preparation of dietary supplements for enhanced viability of these organisms in the human intestinal tract. The objective of the present investigation was to evaluate the effect of incorporation of B. bifidum NDRI and P. freudenreichii subsp. shermanii MTCC 1371 along with L. acidophilus R on the technological and dietetic behaviour of starter cultures and to assess their suitability for the manufacture of probiotic acidophilus milk for infant feeding.
Materials and methods Type of milk
Reconstituted skim milk (SM) [0.5 per cent fat and 7.64 per cent solid non-fat (SNF)] was used for evaluating rate of acid production by starter cultures. Formulated milk (FM) was obtained on fortification of standardised cow milk (2.5 per cent fat and 10.46 per cent SNF) with vitamins A, D and E (Roche Chemicals, Bombay, India) at the levels of 500 IU, 80 IU and 750 mg respectively and addition of 12 per cent sucrose (Misra and Kuila, 1992). Different technological and dietetic attributes of starter cultures were evaluated in both SM and FM.
Starter cultures
Freeze dried cultures of Bifidobacterium bifidum NDRI (National Collection of Dairy Cultures, National Dairy Research Institute, Karnal, India) and Propionibacterium freudenreichii subsp. shermanii MTCC 1371 (Institute of Microbial Technology, Chandigarh, India) were maintained in sterile SM containing 1 per cent dextrose and 0.1 per cent yeast extract as suggested by Misra and Kuila (1991). Lactobacillus acidophilus R (National Collection of Dairy Cultures, National Dairy Research Institute, Karnal, India) was maintained in plain sterile SM.
Pathogenic cultures
Virulent pathogenic strains of Bacillus cereus, Shigella dysenteriae (National Collection of Dairy Cultures, National Dairy Research Institute, Karnal, India), Escherichia coli 03, 018, 078 and Salmonella typhimurium P3(Department of Veterinary Microbiology, West Bengal
University of Animal and Fishery Sciences, Nadia, India) were maintained on nutrient agar slants (Hi-Media, Bombay, India) by weekly propagations and were activated by three successive transfers at 24 h intervals in nutrient broth (Hi-Media, Bombay, India). Analytical techniques
Technological characteristics
Technological attributes of starter cultures were evaluated on the basis of titratable acidity (BIS, 1960), diacetyl and acetoin production (King, 1948), volatile acidity (Hempenien and Liska, 1968) and extent of proteolysis (Hull, 1947).
Dietetic characteristics
Dietetic attributes of starter cultures were evaluated on the basis of lactic acid content (Barker and Summerson, 1941), lactose hydrolysing activity (Citti et al., 1965), antibacterial activity (BSI, 1968) and bile salt tolerance using Man Rogosa Sharpe (MRS) broth (Hoier, 1992).
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Statistical analysis
Results obtained in the present investigation were analysed statistically by the method of Snedecor and Cochran (1967).
Results and discussion Technological characteristics
Titratable acidity. Effect of incorporation of P. freudenreichii subsp. shermanii MTCC 1371 and B. bifidum NDRI along with L. acidophilus R on the rate of acid production, expressed in terms of lactic acid in autoclaved SM and FM, incubated at 37¡1
˚
C, was investigated. Rate of acid production by L. acidophilus R and its association with P. freudenreichii subsp. shermanii MTCC 1371 and/or B. bifidum NDRI were higher in FM than in SM (Table I). Incorporation of B. bifidum NDRI with L. acidophilus R in FM induced an improvement in acid production throughout the incubation. Incorporation of P. freudenreichii subsp. shermanii MTCC 1371 with L. acidophilus R also induced an improvement in the rate of acid production in FM (0.378–1.368 per cent lactic acid) throughout the incubation, however, improvement could be observed after 8 h of incubation in SM (1.116 per cent lactic acid). Conjugated use of P. freudenreichii subsp. shermanii MTCC 1371 and B. bifidum NDRI with L. acidophilus R induced a higher rate of acid production after 4 and 12 h of incubation in SM and throughout the incubation in FM. Because the starter cultures attained the desired level of acidity of 0.6–0.7 per cent as suggested by Foster et al. (1958), an incubation period of 12 h was recommended, keeping in consideration the dietetic characteristics. A minimum incubation period of 12 h for optimum antibacterial activity of B. bifidum and L. acidophilus has been reported (Anand et al., 1984; Prasad and Gandhi, 1987). Incorporation of B. bifidum NDRI and P. freudenreichii subsp. shermanii MTCC 1371 with L. acidophilus R may be suggested for the manufacture of probiotic acidophilus milk for infant feeding.Volatile acidity. The volatile acidity expressed in terms of ml 0.1 N NaOH/50 g curd is depicted in Table II. L. acidophilus R in association with B. bifidum NDRI showed no improvement in volatile acid production in either of the milks, however, significant improvement (p,0.05) could be registered in SM due to its association with P. freudenreichii subsp. shermanii MTCC 1371. Parker and Moon (1982) reported a beneficial synergistic effect on volatile acid production between L. acidophilus and P. freudenreichii subsp. shermanii. Extent of volatile acid production by L. acidophilus R in association with P. freudenreichii subsp. shermanii MTCC 1371 in SM remained unaltered (2.5 ml 0.1 N NaOH/50 g curd) even after introduction of B. bifidum NDRI.
Starter culturesa
Acid production (per cent lactic acid)
TableI. Effect of incorporation of bifidobacterium and propionibacterium on the rate of acid production by Lactobacillus acidophilus Duration of incubation at 37¡1˚C (h) 4 8 12 SM FM SM FM SM FM LA 0.315 0.360 0.585 0.622 1.052 1.126 LA+P 0.261 0.378 0.468 0.783 1.116 1.368 LA+B 0.405 0.513 0.783 0.864 0.865 1.260 LA+P+B 0.333 0.495 0.567 0.990 1.206 1.332 Notes:aAll cultures were inoculated at 1 per cent level individually. SM: skim milk; FM: formulated
milk; LA: Lactobacillus acidophilus; P: Propionibacterium freudenreichii subsp. shermanii; B: Bifidobacterium bifidum
Probiotic
acidophilus milk
Brown and Townsley (1970) reported Bifidobacterium cultures to produce less than 0.05% volatile acidity.
Diacetyl and acetoin content. The diacetyl and acetoin contents along with volatile acids contribute to the characteristic aroma and flavour of the product. Incorporation of P. freudenreichii subsp. shermanii MTCC 1371 with L. acidophilus R induced a significant (p,0.05) improvement in diacetyl and acetoin production, irrespective of the type of milk. However, introduction of B. bifidum NDRI with L. acidophilus R induced an improvement in diacetyl and acetoin production only when cultured in SM (Table II). B. bifidum cultures are reported to produce mainly acetoin while diacetyl is present only in traces (Dolezalek and Plockova, 1981). Conjugated use of P. freudenreichii subsp. shermanii MTCC 1371 and B. bifidum NDRI with L. acidophilus R resulted in a significant (p,0.05) improvement in diacetyl and acetoin production, irrespective of type of milk used. Conjugated use of all these three cultures in the manufacture of probiotic acidophilus milk is recommended.
Proteolytic activity. It is difficult to set certain parameters of selection because proteolysis exerts opposite effects, such as an increase in the digestibility (dietetic characteristic) and a decrease in the consistency or flavour of culture (technological characteristic). The results pertaining to the proteolytic activity of starter cultures, expressed in terms of mg tyrosine/ml are given in Table II. L. acidophilus R alone or in association with P freudenreichii subsp. shermanii MTCC 1371 and/or B. bifidum NDRI had significantly (p,0.05) higher proteolytic activity in SM than in FM. L. acidophilus R in association with B. bifidum NDRI and/or P. freudenreichii subsp. shermanii MTCC 1371 showed no improvement in proteolytic activity in SM, however, a significant (p,0.05) improvement was registered when cultured in FM. Extent of proteolysis by L. acidophilus R in FM was higher when used in association with P. freudenreichii subsp. shermanii MTCC (240 mg tyrosine/ml) than with B. bifidum NDRI (200 mg tyrosine/ml). Results indicate P. freudenreichii subsp. shermanii MTCC 1371 to be more proteolytic in nature than B. bifidum NDRI. Proteolytic nature of B. bifidum (Misra and Kuila, 1991) and propionic acid bacteria (Brendehaug and Langsrud, 1985) has been reported. Conjugated use of P. freudenreichii subsp. shermanii MTCC 1371 and B. bifidum NDRI with L. acidophilus R may be suggested to have a moderate proteolytic activity and to satisfy both technological and dietetic criteria of probiotic acidophilus milk.
TableII.
Effect of incorporation of bifidobacterium and propionibacterium on the technological and dietetic characteristics of Lactobacillus acidophilus Starter culturesa Titratable acidity (per cent
lactic acid) Volatile acidity (ml 0.1 N NaOH/ 50 g curd) Diacetyl and acetoin (ppm) Proteolytic activity (mg tyrosine/ ml) Lactic acid (mg/ml) b-D-gal actosidase activity (mg ONP/ml) SM FM SM FM SM FM SM FM SM FM SM FM LA 1.052 1.126 1.5 1.8 5.0 5.0 440 170 330 220 108 38 LA+P 1.116 1.368 2.5 0.8 9.0 7.0 310 240 320 220 95 33 LA+B 0.855 1.260 1.2 0.5 7.0 4.6 215 200 330 220 85 31 LA+P+B 1.206 1.332 2.5 1.5 10.0 11.0 240 187 330 220 86 44 Notes:aInoculated at 1 per cent level individually and incubated at 37¡1˚C for 12 h. SM: skim milk; FM: formulated milk; LA: Lactobacillus acidophilus; P: Propionibacterium freudenreichii subsp. shermanii; B: Bifidobacterium bifidum
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Dietetic attributes
Lactic acid. Lactic acid production was observed to be significantly (p,0.05) higher in SM (330 mg/ml) than in FM (220 mg/ml) by all starter combinations except for mixed cultures of L. acidophilus R and P. freudenreichii subsp. shermanii MTCC 1371, which produced the same degree of lactic acid (220 mg/ml) in both type of milk (Table II). Lactic acid production by L. acidophilus R in FM remained unaltered either due to incorporation of P. freudenreichii subsp. shermanii MTCC 1371 and/or B. bifidum NDRI. Results indicate disparity in the behaviour of P. freudenreichii subsp. shermanii MTCC 1371 in SM and FM. Lowering of lactic acid content may be due to its utilisation by P. freudenreichii subsp. shermanii MTCC 1371 as reported by Parker and Moon (1982). Incorporation of B. bifidum NDRI and P. freudenreichii subsp. shermanii MTCC 1371 with L. acidophilus R may be recommended for the manufacture of probiotic acidophilus milk for infant feeding without the risk of high acid load. Probiotic acidophilus milk with B. bifidum NDRI may be assumed to have L(+) lactic acid as a major form of lactic acid because in bifidobacteria based fermented milk, dominance of the desired L(+) lactic acid to a level of 90 per cent is reported (Klupsch, 1983).
b-D-galactosidase activity. b-galactosidase (b-gal) activity of L. acidophilus R in association with P. freudenreichii subsp. shermanii MTCC 1371 and/or B. bifidum NDRI was observed to be significantly (p,0.05) higher in SM (85–108 mg ONP/ml) than in FM (31–44 mg ONP/ml). Introduction of P. freudenreichii subsp. shermanii MTCC 1371 or B. bifidum NDRI to L. acidophilus R showed lower b-gal activity with respect to control, irrespective of the type of milk (Table II). Conjugated use of L. acidophilus R, B. bifidum NDRI and P. freudenreichii subsp. shermanii MTCC 1371 in FM induced an improvement in b-gal activity. Presence of b–gal enzyme in L. acidophilus (Fisher et al., 1985), B. bifidum (Premi et al., 1972) and P. freudenreichii subsp. shermanii (Kujawski et al., 1990) has been reported. Conjugate application of all these three cultures is suggested to obtain probiotic acidophilus milk, which would be highly suitable for lactose-intolerant infants.
Antibacterial activity. All starter combinations exhibited variable inhibitory activity against the pathogenic test organisms (Table III). Incorporation of P. freudenreichii subsp. shermanii MTCC 1371 with L. acidophilus R in SM induced an improvement in the antagonism against all the test organisms except for E. coli 078. However, when cultured in FM, an improvement in antagonism against S. typhimurium P3 could only be observed. Incorporation of B. bifidum NDRI also
induced an improvement in antagonism against all test organisms in both SM and
Starter culturesa
Diameter of zone of inhibitionb(mm)
TableIII. Effect of incorporation of bifidobacterium and propionibacterium on the antibacterial properties of Lactobacillus acidophilus S. typhimurium P3 E. coli 078 E.coli 03 E. coli 018 B. cereus S. dysenteriae
SM FM SM FM SM FM SM FM SM FM SM FM LA 6.5 7.0 6.5 7.0 5.5 6.5 5.5 6.0 5.5 6.0 6.5 7.0 LA+P 7.0 7.5 6.5 6.5 6.0 6.5 6.0 6.0 6.0 6.0 7.0 7.0 LA+B 7.0 7.5 7.0 7.5 7.0 7.5 6.5 7.0 6.0 6.5 7.0 7.0 LA+P+B 7.5 8.0 7.0 7.5 7.0 8.0 6.5 7.0 7.0 7.0 7.0 8.0 Notes:aInoculated at 1 per cent level individually and incubated at 37¡1˚C for 12 h. Including the diameter of the well (5 mm). SM: skim milk; FM: formulated milk; LA: Lactobacillus acidophilus, P: Propionibacterium freudenreichii subsp. shermanii; B: Bifidobacterium bifidum
Probiotic
acidophilus milk
FM, however, no difference in antagonism against S. dysenteriae in FM could be observed. Acidophilus milk exhibited antimicrobial activity against 92 per cent Salmonella, 76 per cent Shigella and 67 per cent E. coli (Anon, 1999). Results indicate better antagonism of pathogens by L. acidophilus R when used in association with B. bifidum NDRI than with P. freudenreichii subsp. shermanii MTCC 1371. Amelioration in the antibacterial activity may be attributed to the production of a higher amount of antimicrobial compounds by increased B.bifidum NDRI population in mixed culture with L. acidophilus. R. Khedkar et al. (1994) registered viable cell counts of Bifidobacteria and L. acidophilus to be much higher in mixed culture. Conjugated use of B. bifidum NDRI and P. freudenreichii subsp. shermanii MTCC 1371 with L.acidophilus R exhibited improved degree of antagonism against all test organisms with respect to antagonism registered due to L. acidophilus R alone. Production of bacteriocin-like compounds, designated as MicrogardTM (Al-Zoreky et al., 1993), acidophilin (Shahani et al., 1977) and bifidin (Anand et al., 1984) by P. freudenreichii subsp. shermanii, L. acidophilus and B. bifidum respectively, has been reported. Banina et al. (1998) reported that antagonism of L. acidophilus may be due to lactic acid production rather than hydrogen peroxide or bacteriocin. Exhibition of maximum antagonism against all test organisms suggested conjugated use of P. freudenreichii subsp. shermanii MTCC 1371, B. bifidum NDRI with L. acidophilus R for the manufacture of probiotic acidophilus milk, which would compete with undesirable flora and maintain the normal gut flora of infants.
Bile salt tolerance. The most important criteria for selection of a starter culture for the manufacture of a therapeutic product for infants are its ability to withstand and grow under the conditions prevailing in the intestine. Bile salt tolerance of starter cultures was determined by their capability to grow in the presence of 0.5, 1.0 and 2.0 per cent bile salt within 2 h of incubation at 37¡1
˚
C in MRS broth. Results showed that P. freudenreichii subsp. shermanii MTCC 1371 failed to grow in the presence of 0.5–2.0 per cent bile salt and hence were not bile salt tolerant. However, B. bifidum NDRI and L. acidophilus R had appreciable growth in the presence of 0.5 per cent bile salt. Survival of B. bifidum in the presence of 0.2 per cent sodium tauroglycocholate (Misra, 1988) and L. acidophilus in the presence of 0.15% oxgall (Brennan et al., 1986) has been reported. Shah and Jelen (Misra, 1992) have reported a higher bile salt tolerance of L. acidophilus and B. subsp. than in yoghurt cultures. Survival of L. acidophilus R and B. bifidum NDRI in the presence of bile salt suggested their use in the manufacture of probiotic acidophilus milk for infants with better prophylactic properties.Conclusion
Based upon technological and dietetic characteristics, incorporation of B. bifidum NDRI and P. freudenreichii subsp. shermanii MTCC 1371 along with L. acidophilus R is recommended for the manufacture of probiotic acidophilus milk with enhanced dietetic properties. An inoculation rate of 1 per cent each culture and an incubation temperature of 37¡1
˚
C for 12 h are recommended for the manufacture of probiotic acidophilus milk. Probiotic acidophilus milk may be suitable for normal as well as lactose-intolerant infants and children and possess the capability to maintain the normal gut flora.References
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S. Sarkar can be contacted at: [email protected]
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