SPECIES AFFILIATION OF DAIRY LACTOBACILLI WITH ANGIOTENSIN CONVERTING ENZYME INHIBITORY ACTIVITY

Articles MB SPECIES AFFILIATION OF DAIRY LACTOBACILLI WITH ANGIOTENSIN CONVERTING ENZYME INHIBITORY ACTIVITY T. Stefanova, Z. Urshev, Z. Dimitrov, N. Fatchikova, S. Minkova LB Bulgaricum PLC., R&D Center, Sofia, Bulgaria Correspondence to: Tsona Stefanova E-mail: tsona.stefanova@lbbulgaricum.bg ABSTRACT A group of 72 dairy lactobacilli were evaluated for their ability to produce fermented milk with Angiotensin Converting Enzyme inhibitory activity (ACEIA). The strains were distributed in four clusters: 42 strains with 0-40% ACEIA; 11 strains with 40-50% ACEIA; 13 strains with 50-60% ACEIA and 6 strains with ACEIA over 60%. Ten cultures with the highest ACEIA were selected and their species identification was confirmed according to their carbohydrate fermentation pattern, total cell protein SDS-PAGE profiles and restriction polymorphism of their 16SrDNA (ARDRA). Six of the selected strains proved to belong to Lactobacillus helveticus, two potent ACEIA producing L. helveticus strains were reassigned as L. delbrueckii ssp. bulgaricus, and two other strains were reclassified as L. helveticus and L. delbrueckii ssp. lactis. Keywords: Lactobacilli, ACE inhibitory activity, identification Introduction The inhibition of the Angiotensin-Converting Enzyme (ACE) is a basic approach in the therapy of high blood presure in humans. This enzyme is responsible for the transformation of peptide hormones bradykinin and angiotensin which act as regulators of blood pressure. The inhibitors of ACE are specific peptides, which are the active substance in different medications designed for treatment of high blood pressure. During milk fermentation lactic acid bacteria (LAB) decompose milk proteins to biologically active peptides, including peptides with ACE-inhibitory activity. Among the first reports on such peptides are those of Nakamura et al. (9, 10) and Yamamoto et al. (18, 19) referring to strains of Lactobacillus helveticus used to produce fermented milk with ACE inhibitory activity (ACEIA). The respective peptides with aminoacid sequence of Val-Pro-Pro or Ila-Pro-Pro were found to be produced from beta-casein during fermentation, were not digested by gastro-intestinal enzymes and were absorbed to enter the blood stream (11). However the formation of ACEinhibitory peptides in milk is not a general feature of all LAB. Pihlanto-Leppala et al (12) have studied the ACEIA of whey and casein protein solutions fermented with starter cultures produced by Chr.Hansen Laboratories and Valio Ltd. ACEIA after 6h or 22h fermentation period was not detected either for yoghurt starters or for mixed mesophilic cultures respectively. Therefore to obtain fermented milks containing peptides with ACEIA, LAB with specific features should be first selected as such strains are not commonly met. Shim et al. (15) presented results showing that milk fermented with Lactobacillus casei HY418 had higher (65%) ACEIA than those of milks fermented with various lactobacilli. Strains belonging to the species Lactobacillus delbrueckii ssp. bulgaricus, Streptococcus thermophilus and Lactococcus lactis have been also tested for ACEIA by Gobbetti et al (5), Manisha and Chand (7) and Donkor et al (4). These authors demonstrated that particular strains of L. delbrueckii ssp. bulgaricus (L. bulgaricus) have good potential to generate ACE-inhibitory activity in milk. Among LAB, lactobacilli are the preferred group used in the production of fermented milk with ACEIA. This biological activity is not associated to particular Lactobacillus species, but is rather highly strain specific feature and depends on the particular peptidase activities of the culture. The aim of this work was to select Lactobacillus strains from the LBB culture collection and to clarify the species affiliation of the most potent ACEIA producing cultures. Materials and Methods Strains and growth conditions The target group of the study were strains of Lactobacillus helveticus, L. casei, L. acidophilus and L. bulgaricus, all preserved in the LBB culture collection (LB Bulgaricum PLC, Sofia, Bulgaria). All these strains were initially isolated from home-made fermented milk products. A group of reference strains of the above species were also included. The list of all strains tested for ACEIA is resented in Table 1. Additionally for the purpose of species identification the following reference strains were used: L. acidophilus ATCC4356; L. helveticus ATCC15009; L. plantarum ATCC14917; L. casei ATCC393; L. paracasei ATCC25302; L. delbrueckii ssp. lactis ATCC12315; L. delbrueckii ssp. delbrueckii ATCC9649 and L. delbrueckii ssp. bulgaricus ATCC11842. For all analyses activated strains were inoculated in pasterized 9% reconstituted skim milk and incubated for up to 24 hours at 37 o C. Detection of ACE inhibitory activity (ACEIA) Samples were processed according to Nakamura et al. (10). The ACEIA was determined according to Cushman and Cheung (2) as modified by Nakamura et al. (10). The assay was performed 1250 Biotechnol. & Biotechnol. Eq. 23/2009/2

in a total volume of 300 μl containing 4mM of the substrate (Hip-His-Leu); 4mU of ACE; 300mM of NaCl and 100mM borate buffer (ph 8.3) - 100 mm with or without the addition of processed sample. After exactly 30 min at 37 o C the reaction was terminated with 250 μl 1N HCl and the liberated hippuric acid was extracted with ethylacetate, dried and resuspended in deionized water. The content of hippuric acid was measured Strains from LBB collection studied for ACE inhibitory activity spectrophotometrically at 228 nm. The ACEIA was expressed as percentage of decrease in activity compared to an uninhibited control (0%). The measurement of ACEIA was done in series of ten strains and was repeated at least twice. Determination of the proteolytic activity The proteolytic activity of the strains was determined according to the Hull procedure (6) by measuring the content of Folin TABLE 1 Species Strain Species Strain Lactobacillus helveticus H9 Lactobacillus helveticus NCDO H1 H13 NCDO J4 H25 NCDO 87 H45 NCDO 2 395 H47 NBIMCC h507 H48 NBIMCC 1385 H49 Lactobacillus casei 71a H50 72a H70 A157 H149 C152 H521 C169 Cu40 D Ah D1 Ch D2 Ch1 2465 Dh 2468 Hh 2470 4/135 2471 4/148 2472 4/149 2480 Hv/14 2481 Hvc1 2482 Hv25 Lactobacillus acidophilus L.a. Hv28 L.a.13 Hv83 L.a.14 Hv84 L.a.15 Hv85 L.a.16 H3/n Lactobacillus bulgaricus B5 ATCC 10 797 B26 ATCC 15 809 B35 ATCC 1 114 B77 ATCC 11 977 B128 ATCC 27 558 B146 ATCC 33 409 B230 ATCC 8 018 B241 ATCC 15 009 B337 NBIMCC National Bank for Industrial Microorganisms and Cell Cultures, Bulgaria Biotechnol. & Biotechnol. Eq. 23/2009/2 1251

Clustering of strains according to their ACE inhibitory activity TABLE 2 Number of ACE inhibitory activity in % Lactobacillus studied species I II III IV strains 0-40 % 40-50 % 50-60 % > 60% Lactobacillus helveticus 42 22 6 10 4 - local isolates 28 12 4 8 4 - reference strains 14 10 2 2 - Lactobacillus casei 16 10 3 2 1 Lactobacillus acidophilus 5 2 1 1 1 Lactobacillus bulgaricus 9 8 1 - - Number of strains in different clusters 72 42 11 13 6 Strains selected for their good ACE inhibitory activity TABLE 3 Strain Species Cell number cfu/ml ph Proteolytic activity, mg l -1 Tyr eq ACEIA, % (average) H9 L.helveticus 3.8 E6 3.59 130 58 H25 L.helveticus 1.3 E9 3.93 113 58 H45 L.helveticus 1.9 E9 3.50 148 56 H521 L.helveticus 1.2 E8 4.82 47 60 4/135 L.helveticus 1.3 E9 4.23 96 62 4/149 L.helveticus 1.2 E9 4.78 81 61 Hv25 L.helveticus 1.6 E9 3.75 178 62 Hv28 L.helveticus 2.3 E9 3.48 166 59 2465 L.casei 2.3 E9 4.11 98 66 L.a. L.acidophilus 1.8 E8 3.45 143 70 Species identification of ten dairy lactobacilli with high ACE inhibitory activity TABLE 4 Strain Initial affiliation to Methods for identification New affiliation to species API 50 CH SDS-PAGE ARDRA species H9 L.helveticus L.helveticus L.helveticus L.helveticus L.helveticus H25 L.helveticus L.helveticus L.helveticus L.helveticus L.helveticus H45 L.helveticus L.helveticus L.helveticus L.helveticus L.helveticus H521 L.helveticus L.helveticus L.helveticus L.helveticus L.helveticus 4/135 L.helveticus L.bulgaricus* L.bulgaricus L.bulgaricus L.bulgaricus 4/149 L.helveticus L.bulgaricus L.bulgaricus L.bulgaricus L.bulgaricus Hv25 L.helveticus L.helveticus L.helveticus L.helveticus L.helveticus Hv28 L.helveticus L.helveticus L.helveticus L.helveticus L.helveticus 2465 L.casei L.lactis** L.lactis L.lactis L.lactis L.a. L.acidophilus L.helveticus L.helveticus inconclusive L.helveticus * Lactobacillus delbrueckii ssp. bulgaricus ** Lactobacillus delbrueckii ssp. lactis 1252 Biotechnol. & Biotechnol. Eq. 23/2009/2

phenol reagent-positive aminoacids and peptides in fermented milk using tyrosine as standard. Results were calculated after subtraction of the value obtained for non-inoculated milk and were expressed as mg l -1 tyrosine equivalents. Species identification of strains Ten strains selected as promising for further studies were subjected to identification procedure using contemporary typing methods. The range or carbohydrates fermented by the cultures was determined with the API 50 CH System and the manufacturer s data bank (BioMerieux SA, Marsy l Etoile, France). Total proteins were isolated and separated by SDS- PAGE as described by Tsakalidou et al. (16). Amplified restriction fragments length polymorphism of the 16S-rDNA (ARDRA) was performed according to Miteva et al. (8) using the primers and PCR conditions described by Rodtong and Tannock (13). Results and Discussion The distribution of the 72 tested Lactobacillus strains according to their ability to produce fermented milk with ACEIA is presented in Table 2. The inhibitory activity of every strain was measured at least two times and the averige value was used for classification of strains into four clusters. Low or no activity (less than 40% ACEIA) was demonstrated in 42 cultures (cluster I) of all strains tested. One third of the studied strains had medium ACEIA in the range of 40-60% (clusters II and III). Only six strains (cluster IV) showed activity above 60 %. These included 4 local isolates of L. helveticus, one L. acidophilus and one L. casei according to their initial identification and classification in the LBB collection. After the analysis of ACEIA ten promising strains (all six from cluster IV and four strains from cluster III) were selected for additional studies. Their ACEIA was measured in one and the same experiment as the initial measurements were performed in different series. High values of ACEIA in the range of 56-70% were confirmed for the group of 10 selected strains. The number of viable cells and final ph values at the end of the incubation period showed significant variations among the different strains (Table 3). Strain H521 had especially low acidification rate, compared to the rest of L. helveticus strains which acidified milk bellow ph 4.00. The proteolytic activity of lactobacilli accumulating the highest ACEIA in fermented milk varied in wide range of 80 180 mg l -1 (Table 3). No correlation could be found between the overall proteolysis and the presence of ACEIA in fermented milk. Indeed, ACEIA is attributed to the generation of specific peptides, resulting from the activity of intracellular peptidases. Among several species of lactobacilli L. helveticus and L. delbrueckii ssp. bulgaricus have been shown to have potent aminopeptidase activities in cell extracts (14). The variation in the values of final ph with strains 4/149, 4/135, H521 and 2465 acidifying milk to ph above 4.00, while viable cell numbers remained comparable between the Biotechnol. & Biotechnol. Eq. 23/2009/2 ten strains, suggested the possibility that some of the cultures were not reliably identified using conventional phenotypic and biochemical methods in the period of their isolation about forty years ago. Therefore additional identification procedure was applied combining three methods: carbohydrates fermentation profiles (API tests), SDS- PAGE profiles of total cell proteins and ARDRA. The summarized results from new identification are presented in Table 4. Fig. 1. Total cell proteins patterns of Lactobacillus strains obtained by SDS- PAGE 1. L. casei ATCC393 8. strain 4/149 2. L. delbrueckii ssp. bulgaricus 9. strain 4/135 ATCC11842 10. strain H521 3. strain H25 11. strain L.a. 4. strain H45 12. L. delbrueckii ssp. lactis 5. strain Hv25 ATCC12315 6. strain H9 13. L. helveticus ATCC15009 7. strain Hv28 14. strain 2465 The species affiliation of six of the ten strains was confirmed by the three methods as belonging to L. helveticus. However the carbohydrate fermentation tests of strains 4/149 and 4/135 showed the typical profile of L. bulgaricus. Total cell protein SDS-PAGE patterns of strains 4/149 and 4/135 (Fig. 1, lanes 8 and 9) were identical to those of L. bulgaricus ATCC11842 (lane 2) and clearly different from those of L. helveticus ATCC15009 (lane 13). Digestion of 16S rdna amplified from strains 4/149 and 4/135 with HaeIII (Fig. 2B, lanes 11 and 12) matched the pattern of the L. delbrueckii group of subspecies (Fig. 2B, lanes 6-8) and the pattern obtained with EcoRI narrowed the identification to L. bulgaricus (Fig. 2A, lanes 8, 11 and 12). Therefore former strains L. helveticus 4/149 and 4/135 were reassigned as belonging to the L. bulgaricus subspecies. Two other strains L. acidophilus L.a. and L. casei 2465 also turned out not to be properly identified. The carbohydrate profiles and the total cell protein SDS-PAGE patterns of strain L.a. clearly matched those of L. helveticus ATCC15009 (Fig. 1, lanes 11 and 13). Consequently strain L.a. was reassigned as L. helveticus. However strain L.a. lacked EcoRI restriction sites on its 16S rdna, which disagrees with its affiliation to L. helveticus (Fig. 2A, lanes 2 and 16). Analysis of the complete sequence of the 16S rdna of this strain will be needed for its complete identification. 1253

species identification is especially important when offering a product containing LAB with health promoting properties to provide the consumer with accurate information on the applied culture. Conclusions Our results reavealed that among the dairy lactobacilli preserved in the LBB collection, strains with high ACE inhibitory activity in the fermented milk could be selected. These strains have potential to be used for functional foods development. The selection of two L. bulgaricus strains and one L. lactis strain demonstrating ACEIA comparable to other L. helveticus strains reveales possibilities for diversification of the range of health promoting dairy products using lactobacilli from several species. Fig. 2. ARDRA profiles of Lactobacillus strains obtained by digestion of amplified 16SrDNA with EcoRI (A.) and HaeIII (B.) 1. L. acidophilus ATCC4356 9. strain H25 2. L. helveticus ATCC15009 10. strain H45 3. L. plantarum ATCC14917 11. strain 4/149 4. L. casei ATCC393 12. strain 4/135 5. L. paracasei ATCC25302 13. strain Hv25 6. L. delbrueckii ssp. lactis 14. strain H9 ATCC12315 15. strain Hv28 7. L. delbrueckii ssp. delbrueckii 16. strain L.a. ATCC9649 17. strain H521 8. L. delbrueckii ssp. bulgaricus ATCC11842 18. strain 2465 No evidence of strain 2465 belonging to L. casei species was found. On the opposite, strain 2465 produced the carbohydrate fermentation profile of L. delbrueckii ssp. lactis. SDS-PAGE of the total cell proteins and the ARDRA analyses grouped strain 2465 together with L. delbrueckii ssp. lactis ATCC12315 (Fig. 1, lanes 12 and 14 and Fig. 2). Consequently strain L.casei 2465 was reclassified as L. delbrueckii ssp. lactis. Similar case of unconfirmed species affiliation is reported by Delley et al (3) where three ATCC strains were shown to belong to L. helveticus instead of to L. bulgaricus. Our results confirm once again that for the reliable identification of LAB strains a polyphasic approach should be used. This is also recommended by many authors working in the field of bacterial taxonomy (1, 17). In our study the species affiliation of Lactobacillus strains selected for their property to produce fermented milk with ACEIA was confirmed with molecular methods. Correct REFERENCES 1. Busse H.-J., Denner E.B.M., Lubitz W. (1996) J. Biotechnol., 47, 3-38. 2. Cushman D.W., Cheung H.S. (1971) Biochem. Pharmacol., 20, 1637-1648. 3. Delley M., Mollet B., Hottinger H. (1990) Appl. Environ. Microbiol., 6, 1967-1970. 4. Donkor O.N., Henriksson A., Singh T.K., Vasiljevic T., Shah N.P. (2007) Int. Dairy J., 17, 1321-1331. 5. Gobbetti M., Ferranti P., Smacchi E., Goffredi F., Addeo F. (2000) Appl. Environ. Microbiol., 66, 3898-3904. 6. Hull M.E. (1947) J.Dairy Sci., 30, 881. 7. Manisha N.A., Chand R. (2004) Milchwissenschaft, 59, 4-17. 8. Miteva V., Boudakov I., Ivanova-Stoyancheva G., Marinova B., Mitev V., Mengaud J. (2001) J. Appl. Microbiol., 90, 909-918. 9. Nakamura Y., Yamamoto N., Sakai K., Takano T. (1995a) J. Dairy Sci., 78, 1253-1257. 10. Nakamura Y., Yamamoto N., Sakai K., Okubo A., Yamazaki S., Takano T. (1995b) J. Dairy Sci., 78, 777-783. 11. Ohsawa K., Satsu H., Ohki K., Enjoh M., Takano T., Shimizu M. (2008) J. Agric. Food Chem., 56, 854-858. 12. Pihlanto-Leppala A., Rokka T., Korhonen H. (1998) Int. Dairy J., 8, 325-331. 13. Rodtong S., Tannock G. (1993) Appl. Environ. Microbiol., 59, 3480-3484. 14. Sasaki M., Bosman B., Tan P. (1995) J. Dairy Res., 62, 601-610. 15. Shim J-J., Shin J-H., Lee S-J., Sim J-H., Kim S-K., Baek Y-J. (1999) 6 th Symposium on Lactic Acid Bacteria, Netherlands, Book of Abstracts, J13. 16. Tsakalidou E., Zoidou E., Kalantzopoulos G. (1992) Milchwissenschaft, 47, 296-297. 17. Vandamme P., Pot B., Gillis M., De Vos P., Swings J. (1996) Microbiol. Rev., 6, 407-438. 18. Yamamoto N., Akino A., Takano T. (1994) J. Dairy Sci., 77, 917-922. 19. Yamamoto N., Maeno M., Takano T. (1999) J. Dairy Sci., 82, 1388-1393. 1254 Biotechnol. & Biotechnol. Eq. 23/2009/2