El artículo 145-4

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Nutrition & Food Science Vol. 36 No. 5, 2006 pp. 357-364

EEmerald Group Publishing Limited 0034-6659 DOI 10.1108/00346650610703216

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intestinal epithelial cell prior to its absorption by humans (Miller and Brand, 1980; Hourigan, 1984). In case the quantum of lactose ingested exceeds the hydrolytic capacity of the available intestinal lactase, the undigested portion of lactose is transported to the large intestine, where it increases the osmolarity of the intestinal fluids. Undigested lactose undergoes bacterial fermentation in the colon, generating organic acids, carbondioxide and hydrogen, which along with the large amount of water is drawn into the intestine, are primarily responsible for various symptoms such as bloating, flatulence, abdominal cramps, diarrhoea and loss of appetite (Hourigan, 1984; Hofi, 1990). Lactose maldigestion occurs due to either gastro-intestinal disease or physiological decline in the intestinal lactase activity and may lead to clinical symptoms of lactose-intolerance. Semenza and Auricchio (1995) registered reduction in lactase activity due to digestion of lactase–phlonizin hydrolase molecule by pancreatic proteases at the brush border membrane. Deficiency of enzyme lactase may be of three types (Swaminathan, 1998).

Congenital lactase deficiency

Persons cannot tolerate lactose due to absence of lactase enzyme in the intestine, resulting in accumulation of lactose in the intestine causing abdominal pain and loose motion.

Lactase deficiency in premature infants

This condition occurs in premature infants due to decrease lactase enzyme activity in the intestinal mucosa. Initially, infants cannot utilize lactose efficiently, however they are able to tolerate and digest milk after one month due to increase in lactase activity.

Acquired lactase deficiency

Adults and older children cannot tolerate large amounts of milk due to their non- habitual consumption of milk resulting in low lactase in the intestinal mucosa. Mechanism of lactose digestion

Lactic acid bacteria must survive the gastro-intestinal tract to provide the beneficial effect. Cells of yoghurt cultures contain b-galactosidase as an intracellular enzyme, therefore it is protected during passage through the harsh environment of stomach and is able to reach the small intestine, while still inside the bacterial cells. Permeability of yoghurt cultures is altered, when it comes in contact with bile so that lactose can enter and get hydrolyzed (Gilliland and Kim, 1984). The sensitivity of yoghurt cultures to bile has been proposed as an advantage for lactose digestion, because it increases the permeability of the bacterial cell (McDonough et al., 1987). Shah and Lankaputhra (1997) noted that rupturing of bacterial cells of yoghurt cultures reduced viable counts but the released intracellular b-galactosidase improved the viability of probiotic bacteria such as Bifidobacterium spp. and L. acidophilus which remained above the recommended level of 106cfu/mL. Microorganisms residing in the large intestine made themselves tolerant to lactose through modifications of their metabolic activity (Hertzler and Savaiano, 1996) Efficient utilization of lactose from cultured milk products than in milk may be attributed to improved digestion of lactose resulting from lactase activity of bacteria, stimulation of host’s mucosal lactase activity or slower intestinal transit of cultured milk product compared to milk (Kolars et al., 1984; Gibson and Fuller, 1998).

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Lactase activity in starter cultures

Galactose activity has been demonstrated in many lactobacilli (Mittal et al., 1974) and differs greatly in their lactase activity (Premi et al., 1972; Fisher et al., 1985). Variation in lactase activity of different strains of lactobacillus delbrueckii subsp bulgaricus and streptococcus thermophilus were noted (Gilliand and Kim, 1984) and the later organism possess higher b-galactosidase activity than the former (Lee, 1992). Probiotic cultures such as lactobacillus acidophilus and bifidobacterium, possess lower levels of lactase and being more resistant to bile than yoghurt cultures are less efficacious in helping lactose digestion (Shah and Jelen, 1992). Lactase activity of L. acidophilus strains was reported to vary within a range of 0.5 to 9.5 units (Fisher et al., 1985) and this disparity may be due to micro-heterogeneity in the amino acid composition of lactose (Styrer, 1988). Higher lactase activity of propionic acid bacteria than lactic acid bacteria (Kujawski et al., 1990) suggested their conjugated use during the manufacture of cultured milk products such as dietetic yoghurt (Sarkar and Misra, 1998a, 2001) and Propiono-Acido-Bifido (PAB) milk (Sarkar and Misra, 1998b). Cultured milk products for lactose-intolerant recipients

Better tolerance of yoghurt and acidophilus milk in comparison to milk by lactase non-persistent subjects has been reported (Alm, 1982; Sieber, 2000). A decline in lactose content from 5.26 to 3.19 per cent and an increase in glucose and galactose from 0.05 to 2.11 per cent in yoghurt (Abd-Rabo et al., 1992) and digestion of .90 per cent lactose in small intestine of lactase-deficient subjects due to lactase activity of yoghurt cultures were noted (Streiff et al., 1990). Efficient absorption of lactose by rats from yoghurt containing viable flora (Goodenough and Kleyn, 1976) and a decline in faecal lactase activity in lactase non-persistent human subjects consuming non- pasteurized yoghurt (Pochart et al., 1989) indicated that presence of lactase enzyme and viable flora are necessary for the beneficial effects.

Efficacy of fermented and non-fermented acidophilus milk or bifidus milk is under debate for their benefits for lactose-intolerant subjects. Short-term ingestion of acidophilus milk proved to be not better than milk (Newcomer et al., 1983), and less than yoghurt (Shah et al., 1992; Vesa et al., 1996), however sonication of bacterial cells induced better tolerance by lactase non-persistent subjects and may be ascribed to elevation of lactase activity due to lysis of bacterial cells (McDonough et al., 1987). Kim and Gilliland (1983) reported that addition of a large number of L. acidophilus (2.5 6 106to 2.5 6 108_{cfu/mL) to milk prior to ingestion improved lactose digestion}

and noted a reduction in breath hydrogen due to prolonged consumption of sweet acidophilus milk for 6 days, which may be related to hydrolysis of lactose by L. acidophilus or by lactase in gastro-intestinal tract or reduction in hydrogen producing bacteria (Fernandes and Shahani, 1989). Effect of feeding cultured milk products on breath hydrogen test in humans is shown in Table I.

A number of cultured milk products, namely Antoshka-L (based on bifidobacter- ium), Gnomik – 2 (based on bifidobacterium), Zdorove – 2 (based on L. acidophilus, lactic streptococci, bifidobacterium), Progurt (based on streptococcus diacetylactis or S. cremoris, L. acidophilus and/or B. bifidum), butter milk or yoghurt-like product (based on S. lactis, Leuconostor citrovorum, L. bulgaricus, S. thermoplilus, L. acidophilus or B. bifidum) and PAB milk (based on L. acidophilus, B. bifidum and propionibacterium freudenreichii subsp. shermanii) were recommended for lactose – intolerant infants and children (Schacht and Syrazyski, 1975; Roberts, 1977; Lipatov et al., 1998; Sarkar and Misra, 1998b). Cultured milk products containing

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bifidobacterium and S. thermophilus were tolerated well by infants and the higher level of hydrogen exhaled indicated an earlier bacterial colonization in the digestive tract. (Leke et al., 1999).

Factors affecting lactase activity Microbial growth conditions

b-galactosidase activity of L. acidophilus depends on the growth temperature and pH of medium (Seema et al., 1994). Acid tolerant strains have an advantage in surviving the low pH conditions in the stomach (pH 2.0), where hydrochloric and gastric acids are secreted (Toit et al., 1998). During incubation of yoghurt cultures up to 4 h, b- galactosidase activity reached a maximum value (8 units/g), followed by lowering to a level of 3 units/g, before leveling off. A decrease in enzyme activity between 4–6 h of incubation is due to an increase in titratable acidity (Kilara and Shahani, 1976; Dave et al., 1993).

Microbial viability

Strains of starter cultures must survive the gastro-intestinal tract, which is dependent on buffering capacity of the medium (Conway et al., 1987). Bile-salt tolerance is important for strains to grow and survive in upper small intestine (Toit et al., 1998) and survivality of greater number of bile – resistant lactobacilli strains in gastro- intestinal tract have been reported (Gilliland et al., 1984). A viable population of . 106 cfu/mL is known to exhibit a positive prophylactic effect (Mijacevic et al., 2001). Product processing and storage conditions

Dave et al. (1993) registered higher b-galactosidase activity in dahi made from milk with higher total solids. Higher activity was also noted in formulated milk than in skim milk due to higher total solid content in the former milk (Sarkar and Misra, 1998a). b-galactosidase activity in dahi (Dave et al., 1993) and PAB milk (Sarkar and Misra, 1998b) decreased during refrigerated storage with increasing periods of storage due to shift in pH (Dave et al., 1993). Galvao et al. (1995) noted b-galactosidase activity of 0.58 to 3.3 units in yoghurt, which declined throughout the storage.

TableI. Effect of ingesting cultured milk products on breath hydrogen test in humans Cultured milk product Lactose content (%) Lactase activity (cfu/g) Cell count (cfu/g) Breath hydrogen (ppm) Reference Sweet acidophilus milk 6.30 1427 (mg glucose/dL) 16108 _{28.30 McDonough et al.} (1987) 4.80 0.09 (units/g) – 33.00 Onwulata et al.

(1989) 4.90 0 (mg/h g) 1.16107 _{< 200.00 Savaiano et al.} (1984) Yoghurt 3.60 2.20 (ONPG units) 6.06108 1593 Dewit et al. (1988) 4.20 6.8 (ONPG units) – 9.90 Gilliland and Kim (1984) 4.80 3724 (mg glucose/dL) 2.06108 _{5.40 Mc Donough et al.} (1987) 4.00 0.64 (mg/h g) 3.06108 < 50.0 Savaiano et al. (1984)

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Post-processing treatments

Untreated yoghurt containing live and active flora is tolerated better by lactase non- persistent individuals than pasteurized yoghurt (McDonough et al., 1987; Dewit et al., 1988; Pochart et al., 1989). Pasteurization of yoghurt reduced viable counts from 3 6 108/g to 3.4 6 106/g and lactase activity from 0.64 to 0.07 units/g (Savaiano et al., 1984). Thermization of dahi reduced the lactase activity by 50 to 73.68 per cent and differed with the strains of cultures adopted for dahi manufacture (Sarkar et al., 1992). Conclusion

Possession of b-galactosidase enzyme required for lactose hydrolysis by starter cultures led to their utilization for the manufacture of cultured milk products, suitable for lactose-intolerant individuals. Better tolerance of cultured milk products than milk by lactose-intolerant subjects may be attributed to reduction in lactose content, increase in microbial lactase, stimulation of host’s mucosal lactase activity and slower transit of cultured milk products in comparison to milk. Factors affecting the lactase activity are growth condition survivability of starter cultures, surviability of starter cultures, product manufacturing and storage conditions and post-processing treatments.

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Corresponding author

S. Sarkar can be contacted at: [email protected]

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