Lactic Microflora Present in Liqvan Ewes' Milk Cheese

Abstract

This study aimed to isolate and characterize Lactic Acid Bacteria (LAB) in Liqvan Ewes' milk cheese. A total of 117 Lactic Acid Bacteria were isolated and identified phenotypically. They belonged to 4 genera and 17 species. The dominant LAB found in Liqvan cheese were from the genus Lactobacillus (75.21%) consisted of 70.08% facultatively heterofermentative and 5.12% obligately heterofermentative lactobacillus species. Other isolates were classified as Pediococci (5.12%), Enterococci (5.98%), and Leuconostocs (13.67%). Lb. paracasei subsp. paracasei was the predominant species accounted for 36.75%. Likewise, predominant species of each genus were Lb. paracasei subsp. paracasei, P. pentosaceus, E. faecalis, and Leu. lactis. The preponderance of isolates (86.32%) was referred to be as members of Non Starter Lactic Acid Bacteria (NSLAB).

Keywords:

• Liqvan cheese
• Ewes' milk cheese
• Lactic acid bacteria

INTRODUCTION

Liqvan cheese exemplifies a traditional Iranian white-brined cheese made from raw ewes' milk. It is known as the best cheese in the country for its typical piquant flavor.[1–3] It is classified as a semi soft cheese with a desired sour taste, cream color, high fat content and crumbly texture.[1] The name comes from Liqvan valley near Tabriz city. Its production is limited to a part of the northwestern mountainous area of Iran.[3] Conventionally, coagulation is achieved by natural lamb rennet, and starter cultures are not used. The final coagulum is cut and drained using cheesecloth and pressed. Curing the produced cheeses in brine is carried out in underground rooms dug with access through narrow galleries in width enough for passing just one man.[3] Similarly, the ripening process takes place in these rooms for up to 6 months. The rooms have been used for centuries in this way, and the individual responsible for the creation of these mysterious rooms, and the exact time at which this occurred, remains unknown. The cheese quality entirely depends on different ripening conditions, variations in composition of milk and indigenous microflora. The resultant cheese is well known for its typical sensory characteristics. There have been no previous studies examining the diversity of Lactic Acid Bacteria in liqvan ewes' milk cheese, and understanding what organisms are involved may lead to a better understanding of the development of its desirable organoleptic qualities.

MATERIALS AND METHODS

Cheese Samples

Eight premium quality Liqvan cheese samples were obtained from traditional cheese manufacturers in Liqvan village. The samples were kept refrigerated (4°C) until examination.

Isolation and Identification

An amount of 25 g of each cheese sample was suspended in 225 ml of 0.5% bactopeptone (Difco, Detroit, USA), homogenized by Stomacher® 400 Lab Blender (Seward Medical, London, UK) and serial decimal dilutions were prepared. Diluted samples were plated on MRS agar,[4] and incubated anaerobically at 30±1°C for 48 hrs. Selected bacterial colonies successively transferred 3 times on MRS agar for purification, and checked for catalase and Gram's stain reactions.[5] Gram positive and catalase negative isolates were propagated in MRS broth and stored at −25°C in a 2:1:1 glycerol/MRS broth/reconstituted 20% skim milk powder mixture. When needed, recovery of isolates was undertaken by two consecutive subcultures in MRS broth prior to testing. Each isolate was tested for oxidase, gas production from glucose, growth at different temperatures (10, 15, 40, and 45°C), growth in presence of different NaCl concentrations (2, 4, and 6.5%), Voges-Proskauer, nitrate reduction, polysaccharide formation, haemolysis and ammonia production from arginine.[5] D- and L-isomers of lactate produced by isolates were measured using a previously described colorimetric method.[6] Carbohydrate fermentation patterns of isolates were determined using an API 50 CH kits (bioMerieux sa, Marcy-I'Etoile, France) according to manufacturer's instruction. Finally the isolates were identified using morphological, cultural and biochemical tests according to criteria of Holt;[7] Schleifer and Kilpper-Bälz;[8] Schleifer et al.;[9] Mundt;[10] Devries et al.;[11] Leblanc;[12] Holzapfel et al.;[13] Garvie;[14–15] Björkroth and Holzapfel;[16] Kandler and Weiss,[17] and Hammes and Hertel.[18]

RESULTS AND DISCUSSION

Bacterial isolates were identified based on phenotypic characteristics. Number and percentage of each genus/strain among the sum of isolated LAB has been summarized in Table 1.

Table 1 LAB isolated from Liqvan cheese

Genus/Species	n ^a	% ^b
Facultatively Heterofermentative Lactobacilli	82	70.08
Lb. plantarum	14	11.96
Lb. paracasei subsp. paracasei	43	36.75
Lb. casei	12	10.25
Lb. rhamnosus	2	1.70
Lb. coryniformis subsp. coryniformis	1	0.85
Lb. curvatus subsp. curvatus	4	3.41
Lb. bifermentan	3	2.56
Lb. pentosus	3	2.56
Obligately Heterofermentative Lactobacilli	6	5.12
Lb. brevis	5	4.27
Lb. fermentum	1	0.85
Pediococci	6	5.12
P. penctosaceus	3	2.56
P. acidilactici	2	1.70
P. parvulus	1	0.85
Enterococci	7	5.98
E. faecum	4	3.41
E. faecalis	2	1.70
E. durans	1	0.85
Leuconostoc	16	13.67
Leu. lactis	16	13.67
Total	117	100
^a: Number of isolates; and ^b: percentage of genus/strains among the sum of isolated LAB.

The Gram positive and catalase negative organisms were divided into two morphological groups. These were coccal or ovoid forms and rods. Each group was then subdivided into homofermentative and heterofermentative by examining their fermentation products. Heterofermentative activity was determined by the ability of strains to produce carbon dioxide as a fermentation product from glucose. The majority of LAB isolated from Liqvan cheese belonged to Lactobacilli (75.21%). Facultatively heterofermentative (FHL) and obligately heterofermentative lactobacillus species comprised 70.08% and 5.12% of the isolates respectively.[17–19]

Of the isolates, eighty two were FHL (70.08% of total isolates). Eight species were characterized in this group. The strains that fermented ribose, mannitol and melezitose, but not rhamnose, grew at 15°C and formed L-Lactic acid, were identified as L. paracasei subsp. paracasei (43 strains). Strains that fermented ribose and gluconate, but not glycerol and xylose, grew at 15°C, but not at 45°C, formed DL-Lactic acid and catabolised α-methyl-D-mannoside, were identified as Lb. plantarum (14 strains). The strains that did not ferment ribose and mannitol, grew at 15°C and 45°C and formed L-Lactic acid, were identified as L. casei (12 strains). Four strains that did not produce ammonia from arginine, fermented ribose, but not mannitol, xylose and melibiose, grew at 15°C, but not at 45°C and formed DL-Lactic acid, were identified as L. curvatus. The strains that fermented ribose, mannitol, but not melezitose, grew at 15°C, but not at 45°C and formed L-Lactic acid, were identified as Lb. bifermentan (3 strains). And finally, the strains that fermented ribose, gluconate, glycerol and xylose, grew at 15°C, but not at 45°C and formed DL-Lactic acid, were identified as Lb. pentosus (3 strains). The strains that fermented ribose and rhamnose grew at 15°C and 45°C and formed L-Lactic acid seemed to be Lb. rhamnosus (2 strains). The strain that did not ferment ribose, but mannitol, grew at 15°C, but not at 45°C, formed DL-Lactic acid (more D isomer than L isomer), were identified as Lb. coryniformis subsp. coryniformis (one strain).[18] Of Lactobacilli, six belonged to obligately heterofermentative group (5.12% of total isolates). The species identified consisted of 5 Lb. brevis, and one Lb. fermentum. The strains that fermented ribose, galactose and melibiose, but not mannose, melezitose and gluconate, grew at 15°C, but not at 45°C were identified as Lb. brevis (5 strains). The strain that did not ferment gluconate or grow at 15°C but did so at 45°C was identified as Lb. fermentum (one strain).[18]

FHL form a significant portion of the NSLAB community in most cheese varieties during ripening.[20] In the case of raw milk cheeses, FHL are mainly derived from the cheese milk.[20] They usually increase from a low number in fresh curd to dominate the microflora of ripened cheese.[21] Non starter Lactobacilli can easily tolerate the hostile environment of cheese typically characterized by low pH, low moisture content, high salt concentration, and nutrient deficiency.[22] Non starter lactobacilli have a wide range of proteolytic and lipolytic activities. Proteolytic and lipolytic products contribute to and/or act as precursors for flavour development in cheese.[23]

On a negative note, some species of obligately heterofermentative lactobacilli can produce some end products contributing to texture and off-flavour defects, if present at adequate levels.[23–24] Lactobacilli usually grow as a heterogeneous community in the cheese matrix.[25] The most frequently strains of facultatively and obligately heterofermentative lactobacilli found in raw ewes' milk cheeses include; Lactobacillus paracasei subsp. paracasei, Lb. plantarum, Lb. curvatus, Lb. rhamnosus, Lb. coryniformis, Lactobacillus casei subsp. casei and Lb. brevis.[25–33]

Of the coccal/ovoid isolates, 16 were Leuconostoc. Heterofermentative activity and failure to produce ammonia from arginine were characteristics that allowed Leuconostoc to be distinguished from the other coccal isolates. They were also differentiated from morphologically similar heterofermentative lactobacilli in that they produced no ammonia from arginine and formed D-, but not DL-lactic acid from glucose. All 16 Leuconostoc were identified as Leu. Lactis. They gave a positive Voges-Proskauer test and did not hydrolyze esculine; however they fermented lactose and sucrose. These isolates also did not ferment ribose, cellobiose, trehalose, mannitol and xylose.[14,16,34]

Technologically, Leuconostocs are not main producers of lactic acid in milk and dairy products. Particular strains of Leuconostocs (Leu. mesenteroides subsp. cremoris and Leu. lactis) play a significant role in developing flavour and aroma in certain varieties of cheese.[34–35] They typically contribute to the production of flavour compound diacetyl through carbohydrate heterofermentation in associative growth with some other LAB.[34] Cogan et al.[35] studied 4379 lactic acid bacteria from European artisanal dairy products including some cheeses; they identified about 10% of the strains as Leuconostoc spp. Previously Leu. lactis was isolated as the predominant Leuconostoc from some ewes' milk cheeses.[25,33]

Of the isolates of homofermentative cocci, seven were characterized as Enterococcus by their ability to grow at 10°C and 45°C, as well as their ability to grow in the presence of 6.5% NaCl and at pH 9.6 without any tetrad formation becoming evident. In addition, these homofermentative coccal isolates produced ammonia from arginine, but not carbon dioxide from glucose. There were four E. faecalis, two E. faecium and one E. durans identified. The E. faecalis was differentiated from E. faecium and E. durans by its ability to both reduce triphenyl tetrazolium chloride (TTC) and produce acid from sorbitol. In addition, E. faecalis produced acid from D-arabinose and mannitol, which is not observed for E. faecium. The strain identified as E. durans was unpigmented and did not reduce TTC. Furthermore, it didn't metabolize melibiose, saccharose, mannitol and raffinose.[7–8,10–12]

Enterococci especially E. faecalis and E. faecium are frequently associated with the intestines of human and animals, and when found in food or water suggests faecal contamination.[36] These species are also the most common enterococcal species found in cheese. Enterococci occur as NSLAB in variety of cheeses, especially raw ewes' milk cheeses manufactured in Mediterranean and Middle Eastern countries.[25,37–44] Enterococci can survive and grow in the harsh environment of cheeses with high salt content and low pH.[45] They are likely to originate from the raw milk, and are considered to play an important role in cheese ripening and aroma development due to their proteolytic and lipolytic activities and diacetyl production.[46] It has been reported that the presence of the enterococci in cheese positively affected the growth of other NSLAB, increased the proteolytic indices and positively affected organoleptic properties of the cheese.[47] Furthermore, Enterococci produce bacteriocins that inhibit foodborne pathogens in cheese.[46,48]

Pediococci were differentiated from other isolated homofermentative cocci on the basis that tetrads are formed during cell division. Pediococci constituted 6 isolates that were classified into four species. Two strains did not produce carbon dioxide from gluconate, fermented rhamnose and D-arabinose but not maltose and grew at 48°C, were identified as P. acidilactici. Whereas, P. penctosaceus (3 strains) fermented maltose and did not grow at 48°C. One isolate was identified as P. parvulus. This isolate was characterized by its ability to ferment rhamnose, D-arabinose and galactose, but not ribose and lactose and to grow at pH 7.0 and 35°C.[7,13,15]

The role of pediococci in dairy products is not as important as that of Lactobacilli, Lactococci, Streptococcus thermophilus or certain Leuconostocs.[13] Pediococci have been occasionally involved in ripening and flavor production of some cheeses.[49] They have been isolated as NSLAB from raw ewes' milk cheeses such as Kurdish,[25] Batoz,[28] and Manura,[27] where they may facilitate the cheese making process via their metabolic activities, especially proteolytic activity.[50–51] P. acidilactici and P. pentosaceus are the most frequent species and important members of NSLAB group and are involved in the maturation and ripening of cheeses.[20 ^,52 52–54] Moreover, P. acidilactici and P. pentosaceus produce a wide range of pediocins as biopreservatives that can inhibit some harmful bacteria in cheese.[13]

With the exception of the Leuconostoc, the LAB isolated in this study (86.32%) are members of Non Starter Lactic Acid Bacteria (NSLAB). NSLAB contribute to flavour development.[20,55] Furthermore, among isolates, there were some potential probiotic strains including Leu. lactis, Lb. plantarum, Lb. casei, Lb. rhamnosus, Lb. fermentum, E. faecalis, E. faecium, and P. acidilactici, however they need to fulfill requirements in this way.[56]

CONCLUSIONS

Liqvan cheese is considered to be a supreme product for its flavour and other sensory characteristics. Its qualities are linked to the type of milk (ewes' milk), absence of heat treatment and most importantly, the desired metabolic activities of indigenous microflora of milk during cheese ripening. A wide diversity of LAB mainly Lactobacilli was found to be present in the cheese. Up to 86.32% of all isolates belonged to NSLAB. This diversity implies that LAB may give various attributes to cheese. In addition, among the isolates, there were potential probiotic strains that may contribute to consumer health. Further testing of these naturally occurring bacteria may provide new strains for dairy fermentations.

ACKNOWLEDGMENT

The authors wish to thank Mrs. Rosalind Smith for her language assistance in this paper.