Active microflora Swiss cheese of Armenia

   
    A b s t r a c t. Sergei Rosen. Active microflora of Swiss cheese, manufactured in Armenia.
 
    It was establushed, that microflora of Swiss cheesee manufactured in Armenia included Lactobacillus salivarius,  Lb. lactis, Lb. plantarum, Lb. casei, Streptococcus faecalis, Str. bovis, Propionibacterium acidipropionici   and   Pb. jenseni. The coccus forms were found only in curds. It was demonstrated that local Swiss cheese was characterized by significant amounts of nonprotein nitrogen,  determining the "cheese flavour", and that the specific organoleptic  properties can be attributed to the presence of acetic, propionic and butyric acids and partly to diacetyl and acetaldehyde, the content of which in cheese in several times higher than the taste threshold values of these substances. For the first time the "aroma coefficients" have been calculated for the cheese manufactured in this region. It was found that Lb.lactis, Lb. salivarius, Lb. plantarum, Str. faecalis, Str. bovis, Leuconostoc  paramesenteroides, Pb. acidipropionici  belong to species with the highest ecological potential in the mentioned bacterial groups and are most widely distributed in  indigenous microflora. The experiments with starters  constituted with these species of lactic acid and propionic acid bacteria with enhanced proteolytic and aroma forming activity accelerated the ripening of cheese and improved cheese flavour.

    P r e f a c e.  The aim of the present study was to compare the specific composition of the active microflora of Swees cheese made in the Armenia with the bacterial flora of the same area, with the view of including the most widespread species for Swiss cheese and to elaborate a biological criterion for the selection  of strains offering advantages for the manufacture of Swiss cheese.
   
    Though the flavour compounds of Swiss cheese have been fairly investigated, it was not determined how many times the content ofeach of these compounds exceeded its own taste threshold, i. e. the minimum amount when it can be detected organoleptically/ In the present investigation  cheese was characterized for the first time by the sum of aroma coefficients with the aim to use it as a criterion for the evaluation of cheese according to its flavour.

    Finally, on the basis of these developments, a starter should be combined and tested for the manufacture of  Swiss cheese.

    Swiss cheese in a natural habitat for Lb. helveticus, Str. thermophilus, Pb. freudenreichii (Rogosa, 1974; Deibel and Seeley, 1974; Moore and Holdeman, 1974). Recent data mention also Lb. lactis, Lb. casei, Lb. plantarum, Lb. fermentum, Str. faecalis, Str. bovis,  Str. lactis, Str. cremoris and Pb. shermanii as obligate bacterial species which take part in the ripening of Swiss cheese (Munch et al., 1981). Obviously these are cumulative data, and in different areas of the word the bacterial flora of this cheese may vary according to local conditions.

     M a t e r i a l  a n d   m e t h o d s.

     The specific  bacterial composition of dairy products, higher plants and the faeces of breast fed babies in the Armenian Republic was investigated during the years 1960-1986 (samples Nos. 5-234; strains 639-4690, 6001-6073).

     Table 1  summarizes the data concerning the cheese samples from which strains were isolated. Nos, 31 and 103 are curd samples (1 day), Nos, 33, 34, 203 and 214 - samples  of ripe cheese (6 months).

     Table 1: BACTERIAL SPECIES ISOLATED FROM SWISS CHEESE SAMPLES   
Sample 31, 1966-I, Kalinino; Streptococcus faecalis 1724-1725, 1727-1728, 1731, 1798, 1829-1830; Lactobacillus lactis 1660, 1662, 1726, 1801-1803

Sample 33, 1966-I, Zalka (Georgia); Streptococcus bovis 1570; Lactobacillus lactis 1580, 1586; Lactobacillus salivarius 1579, 1583, 1588, 1590

Sample 34,1966-1,Bazarchay; Lactobacillus salivarius 1615, 1618, 1621,1622, 1628; Lactobacillus lactis 1617, 1619,1623, 1624, 1625, 1626, 1630-1633, 1633, 1635

Sample 54,1967-IX,Kalinino; Lactobacillus casei 2323, 2328-2331;Lactobacillus lactis 2335-2337; Lactobacillus plantarum 2324

Sample 91,1969-VI,Kalinino; Streptococcus faecalis 3190

Sample 101, 1973-II, Bazarchay; Streptococcus faecalis 3245, 3349-3350,3361, 3363, 3369, 3371

Sample 103, 1973-II, Bazarchay; Streptococcus faecalis 3302

Sample 203, 1982-I, Bazarchay; Propionibacterium acidipropionici 6001-6024

Sample 214, 1983-I, Bazarchay; Propionibacterium jensenii 6071-6073

    A medium containing hydrolyzed milk      supplemented with 1% lactose, 0,5% yeast autolysate and 0,5% sodium chloride was used for the isolation of lactic acid bacteria. Incubation was carried out at 37-40 grad. C. The medium for propionibacteria was constituted of 20 ml of 60% solution of sodium lactate, 30 g peptone, 10 ml yeast autolysate and 10 g agar in 1000 ml water, pH 7.3.

    The strains were identified according Rogosa, 1974; Garvie, 1974; Deibel and Seeley, 1974; Moore and Holdeman, 1974; Cummins and Johnson, 1981.

    The identification was carried out according tj the following tests and methods. Staining according to Gram; mobility and relation to oxigen (stab inoculation in 0.1-0.2% hydrolyzed milk agar); spores; pigment; catalase (3% solution of H202); nitrate reduction (Rogosa medium, nitrate determination by the Griss-Ilossa reagent); lactic acid (titration with 0.1 N alcali); form and disposition of cells (staining with methylene blue, microscope 90 x 10); gas from glucose (Gibson-Abdel Malek method); carbohydrate fermentation (Orla-Jensen medium as modified by Palladina:peptone 0,5%, yeast autolysate 2.5%, Na2PO4 0.1%, KH2PO4 0.1%, MgSO4 0.1%, carbohydrate concentration 0.5-1.0%, incubation 7 days, sterilization by Seitz filtration, titration with 0.1 N alcali in presence of phenolphtalein,esculin and hippuric acid used as carbohydrates); growth at 10 grad. C and 15 grad. C 10 days, and at 40, 45 and 50 grad. C during 1 day folloved by pH determination or titration; 10-15 grad C in refrigerator with regulated temperature, 40-50 grad. C - in thermostate). Most of the references according to Skerman (1969).

    The Gram-positive, asporogenic rods failing to form gas on glucose were attributed to streptobacteria if growing at 15 grad. C,otherwisse they were considered as thermobacteria, streptobacteria capable 0f fermenting raffinose, were attributed to Lb. plantarum, if not, to Lb. casei. The thermobacteria capable of fermenting lactose, sacharose, mannitol and maltose but failing to ferment salicin were attributed to Lb. salivarius. The thermobacteria fermenting lactose, maltose, saccharose and salicin but unable to ferment mannitol were attributed to Lb. lactis.

    The Gram-positive cocci unable to form gas on glucose but growing at pH 9,6, at 10 grad C and 45 grad C, in the presence 6.5% NaCl and capable of reducing milk containing 0,1% methylene blue were attributed to enterococci. Gram-positive cocci showing negative reactions against all these tests apart from growth at 45 grad. C were attributed to streptococci. Enterococci capable of using arginine but using arabinose very   weakly were      attributed to Str. faecalis (according Deibel, Seeley, 1974 this species does not use arabinose at all). Streptococci capable of splitting starch,  fermenting lactose and growing in the presence of 40% bile were attributed to Str. bovis. Such strains were also attributed to this species according to the key proposed by one of the authors (Ter-Kazarian and Ter-Simonian, 1986) for the identification of species belonging to streptococci^ these strains grow on blood agar containing 40% bile, they use lactose, do not use ribose and their growth is stimulated under 5% CO2.

    Anaerobic to aerotolerant Gram-positive asporogenic nonmotile rod capable of clotting milk, reducing nitrates, forming catalase and gas, growing in the presence of 20% of bile, but unable of liquefying gelatine or forming indole and failing to induce haemolysis were attributed to propionibacteria. Saccharose and maltose fermentation and nitrate reducton were  attributed to Pb. jenseni.

    The ecological potential was determined as the sum of positive tests, expressed in per cent of the set of properties investigated for all species og a given genus and presenting an ecologic value. The total amount of fermented carbohydrates, splitted biopolymers and tolerance tests are determined for each species,

    The following properties for several genera were selected according Rjgjsa, 1974; Garvie, 1974; Deibel and Seeley, 1974; Moore and Holdeman, 1974.

    STREPTOCOCCUS (table 14.6< 14.7 and text): arabinose, glycerol, maltose, raffinose, sucrose, sorbitol, trehalose fermentation; hydrolyzing activity (starch, inulin, hippurate), and all the tolerance tests (growth at 10 and 45 grad. C, 6,6% NaCl, 40% bile,pH 9.6, methylene blue reduction and thermoresistance), a total of 17 tests.

    LEUCONOSTOC (table 14.8): amygdalin, arbutin, cellobiose, fructose, galactose, lactose, maltose, mannitol, mannose, melibiose, raffinose, salicin, sucrose, trehalose, xylose, aesculin, citrate, malte fermentation; dextran formation; lithmus milk reduction, growth in presence of 3.0 and 6.5% NaCl, at 37 grad. C, pH 4.8 and 6.5, a total of 25 tests.

     LACTOBACILLUS (table 16.1): amygdalin, arabinose, cellobiose, fructose, glucose, lactose, maltose, mannitol, mannose, melezitose, melibiose, raffinose, rhamnose, ribose, salicin,sorbiyol, sucrose, trehalose, xelose, aesculin, a total 20 tests.

     PROPIONIBACTERIUM: amygdalin, arabinose, cellobiose, erythritol, aesculin, fructose, galactosem glycerol, glycogen, inositol, inulin, lactose, maltose, mannitol, mannose, melezitose, melibiose, raffinose,rhamnose, ribose, salicin, sorbitol, sorbose,starch, sucrose, trehalose, xylose fermentation; gelatine liquefacien, nitrate reduction, catalase, a total 30 tests. All superscripts for this genus (tables 17.10 and 17.11) as well as symbols "d" and "v" are expressed as "+_" and considered as half a unity.

     The proteolytic activity of the strains was determined by formol titration; the same in cheese - by the Kjeldal method. The carbonyl compounds and volatile fatty acids (the latter after esterification) were determined by gas chromatographic headspace analysis (Hach enberg and Schmidt, 1977) on "Chrom-3" (CSSR).

     The flavour coefficients indicating how many times the concentration of a given substance exceeds its "taste threshold" were calculated according to Gorbatova (1984).

     R e s u l t s

     The more general pattern of the bacterial flora of Swiss cheese made in Armenia is summarized in Table 1. The isolated species of bacteria belonged to the following species: Lactobacillus lactis  and Lb. salivarius Lb. casei and Lb. plantarum Streptococcus faecalis Str. bovis ); Propionibacterium acidipropionici and Pb. jensenii (propionibacteria). Certain strains  identified as Lb. lactis according to Pederson (1957) were reidentified as Lb. salivarius according to Rogosa (1974), Lb. salivarius, Pb. acidipropionici and Pb. jensenii were found to be specific for this region. Presence of Str. faecalis and Str. bovis is pecular to Swiss cheese manufactured in this region.   

    The isolated species of lactic acid bacteria were investigated for their proteolytic activity and their ability to accumulate aromatic substances in milk and cheese (Table 2).

    Tablt 2. FLAVOUR FORMING SUBSTANCES IN MILR AND CHEESE BY LOCAL LACTIC ACID BACTERIA
                (mg per 100 ml of milk)

Lactobacillus lactis: nonprotein nitrogen 9.7; acetic acid 18, propionic acid 2.3, butyric acid 0.6; diacetyl 0.04, acetaldegyde 0.08.

Lactobacillus salivarius: nonprotein nitrogen 10.6, acetic acid 4, propionic acid 1.2, butyric acid 2.2, diacetyl 0,09, acetaldehyde 0.04.

Lactobacillus plantarum: nonprotein nitrogen 8.0, acetic acid 15, propionic acid 0.3, butyric acid 0.1, diacetyl 0.26, acetaldehyde 0.00.

Enterococcus faecalis: nonprotein nitrogen 6.9, acetic acid 13, propionic acid 0.1, butyric acid 1.4, diacetyl 0.80, acetaldehyde 0.12.

Streptococcus bovis: nonprotein nitrogen 4.2, acetic acid 12, propionic acid 0.4, butyric acid 0.7, diacetyl 0.70, acetaldehyde 0,01.
Starter, including all the mentioned species (mg per 100 g of cheese): nonprotein nitrogen 777.0, acetic acid 38, propionic acid 68.0, butyric acid 16.0, diacetyl 0.01, acetaldehyde 0.30.

Taste threshold: acetic acid 2.2, propionic acid 5.0, butyric acid 2.5, diacetyl 0.001, acetaldehyde 0.12.

Aroma coefficient: acetic acid 17, propionic acid 14, butyric acid 6, diacetyl 7, acetaldehyde 3.

    This table shows at the same time how many times the content of a particular aromatic substances exeeds its taste threshold. These data   allow to calculate the flavour coefficients for each aromatic substance. These data provide, for the first time, the characteristics of Swiss cheese manufactured in this region. The data concerning the ability of different species of lactic acid bacteria isolated from the local microflora to accumulate aromatic substances in milk were partly published earlier (Ter-Kazarian et al., 1978).

    Table 3 shows the results of the identification of lactic acid and propionic acid bacteria constituting the microflora of the Armenian  Republic, including data from Swiss cheese.


    Table 3. THE  OCCURENCE OF SPECIES OF LACTIC ACID AND PROPIONIC ACID BACTERIA IN THE ARMENIAN SSR (samples nos. 5-234, 1960-1986, quantity of isolated strains)

ENTEROCOCCUS: Ec. avium 14, Sc. faecalis 515, Sc. faecium 36.Sc. uberis 30.

STREPTOCOCCUS: Sc. bovis 52, Sc. cremoris 34, Sc. lactis 44.

LACTOBACILLUS:  Lb. casei 107, Lb. helveticus 44, Lb. fermentum 5, Lb. lactis 224,     Lb. plantarum 215, Lb. salivarius 120, Lb. xylosus 12.

LEUCONOSTOC: Lc.  paramesenteroides 20.

PROPIONIBACTERIUM: Pb. acidipropionici 24, Pb. jensenii 3.

    Table 4 shows the figures of the ecological potential of bacterial species already included in starters, or which are of interest for their  future use.

     Table 4: ECOLOGICAL POTENTIAL OF LACTIC ACID AND PROPIONIC ACID BACTERIA,      PRESENTING TECHNOLOGICAL INTEREST

LACTOBACILLUS:  Lb. acidophilus 55, Lb. casei 70,  Lb. helveticus 10,                Lb. lactis 30, Lb.plantarum 88, Lb. salivarius 58.

STREPTOCOCCUS:  Sc.  bovis 55, Sc. cremoris 18, Sc. lactis 42, Sc. salivarius ssp. thermophilus 21.

ENTEROCOCCUS: Sc. faecalis 79, Sc. faecium 74.

LEUCONOSTOC:  Ln. cremoris 12, Ln. dextranicus 52,  Ln. lactis 37,
Ln. mesenteroides 66,    Ln. paramesenteroides 56.

PROPIONIBACTERIUM: Pb. acidipropionici 72, Pb. freudenreicherii ssp. shermanii 42,
Pb. freudenreichuu 33,  Pb. jensenii 53, Pb. thoeni 56.

    From table 4 it can be seen that Lb. salivarius, Ec. faecalis, Lb. plantarum,     Sc. lactis, Lc. paramesenteroides, Pb.     acidipropionici present the highest potential among the representative groups.

    With the aim of increasing the starter activity and starter resistance to a great number of unfavourable conditions starters should be constituted with the species of higher ecological potential.

    The following strains of local species of lactic acid and propionic acid bacteria presenting proteolytic and aroma forming abilities were selected and used in our laboratory in starters for Swiss cheese:  Lb. salivarius 1588, Lb. plantarum 2204, Lb. lactis 2472,2955,  Ec. faecalis 1987, 2005, Pb. acidipropionici 6010. The results presented in Table 5, show that the use of the supplemented starter stimulated  the intensity of proteolysis in cheese, raised the levels of acetic, propionic and butyric acids, which determine the taste of cheese.

 Table 5: CHEESE RIPENING AS INFLUENCED BY A STARTER INFLUENCING  BACTERIAL SPECIES FROM THE AREA OF PRODUCTION (mg per 100 g)

Non protein nitrogen: control 550,0; experemental 777,0.

Acetic acid: control 156.0; experimental 198,0.

Propionic acid: control 86.7; experimental 121.2.

Butyric acid: control 11.5; experimental 17.1.

Acetaldehyde: control 0.013; experimental 0.007.

Diacetyl: control 0.009; experimental 0.007.

Points for taste and aroma: control 39.0; experimental 40.8.

Lentht of ripening period (months): c0ntrol 6.0; experimental 4,5.

    Consequently the ripening period of cheese was considerably reduced (4.5 month instead of 6), this fact presenting a  definite  economic advantage. It was possible to compare the improvement of cheese quality in scores for flavour, with the increase of the aroma coefficients.

D i s c u s s i o n

     Apparantly, the most widespread organisms are those which are the best adapted to their environment. The study of the specific composition of the local microflora indicated that many widespread species as Ec. faecalis and Sc. bovis, Lb. lactis and  Lb. plantarum, Pb. acidipropionici are present in Swiss cheese, the less widespread species being Lb. casei and Pb. jensenii. The starter constituted wity the most common species,  contributed to the intensity 0f proteolysis, the accumulation of flavour compounds, the improvement of cheese flavour and the reduction of the ripening period. These positive changes indicate that the use of starters containing the mentioned strains is worthwhile. The most common species constitute a resource of strains for dairy starters and other biotechnological  applications.

R e f e r e n c e s

Cummins C.S. and Johnson J. L., 1981. Genus Propionibacterium. In Prokaryotes, v.2 pp. 1894-1902. Berlin and al.: Springer Verlag.

Deibel R.H. and Seeley H.W., 1974. Genus Streptococcus. In Bergey's manual of determinative bacteriology, pp. 490-509. Baltimore: William and Wilkins.

Garvie E. J. 1974. Genus Leuconostoc, pp.510-513. In Bergey's manual.

Gorbatova K.K., 1984. Biochemistry of milk and dairy products. Moscow: Pischevaya promyshlennost' (in Russian).

Hachenberg H. and Schmidt A.F., 1977. Gas chromatographic headspace analysis. London et al.: Heyden.

More W.E.C., Holdeman L.V., 1974. Genus Propionibacterium, pp. 633-641. In Bergey's manual.

Munch G.-L. et al., 1981. Mikrobiologie tierischer Lebensmittel. Leipzig: VEB Fachbucherverlag.

Pederson C.S., 1957. Genus Lactobacillus. In Bergey's manual.

Rogosa M. Genus Lactobacillus, pp. 576-593. In Bergey's manual.

Skerman V.B.D., 1969. Abstracts of microbiological methods.

Ter-Kazarian  S.Sh., Saakian R.V., Sarkisian V.K., Ioanisian T.A., Kharatian V.G., Sagoian A.S. and Chobanian M.K., 1978. Flavour compounds produced by lactic acid bacteria during cultivation in milk, pp. 285-286. In 20th International Dairy Congress, Paris