boa riia l of liu try desen rr/i ( 100.

3) 60 2ti3—2ti7 h*ri nteil iii €‹'reriï 13r tia in 2ö3

Microflora present in kefir grains of the Galician region (North-West

of Spain)
Bv /l.UIS ANOULO, EUS DO hOPET AND CESAR LEMA
1Ii ‹' robiologlrt , I“ooultad de Giencias, Llniversidod de Viqo, A p‹irta,do 874, 36200 Vito,

’lie kefir grain is a s3unbiotic association of yeasts and lactic acid bacteria (T AB)
is hich c‹iuses an acid—alcoholic fermentation of milk. This inicr oflora is embedded in
‹i resilien I polysiiccliaride inatri.x called kefiran composed of branched chains of
glucose iiiid giilactose as the result of the microbial metabolism of milk lactose (Isa
Riviére ef u/. 1967 ; 3larshall ct of. 1984). ’The grains develop under certain physical
and chem iciil conditions which inhibit the development of most microorganisms,
suggesting that only a small part of their microbial composition is specific but not
constiwlt. These differences in microbial composition may be explained by the
different techniques employed during processing and/or the different sources of the
kefir gi‹iins (I3emeter, 1969 ; La Riviéi’e, 1969 ; Vayssier, 1978).
’the presence of different yeast species and within this category the predominance
of those species that are unable to fei ment lactose (lactose-negative), as opposed to
liictose-positi ve ,yeasts, has already been described (\*an Uden & Assis-Lopez, 1955 ;
lkosil‹ou ski, 11)77 ; Rosi, 1978 n). In the I AB group the most frequently mentioned
genus is 1>oclobcir ill.its, which includes both homofermentative and hetero-
ferrnentative species, the species predominance depending on the origin of the kefir
gr tins beiiig studied (La Rivière ef o,l,. li)67 ; Kooiman, 1968 ; Vayssier, 1978 ; Kandler
cfi Jû uniitli, 1583).
ftosi (1978 6) iind Rosi & Rossi ( 1978) describe the presence within the grains of’
other LAH species, such as fiacfococcus spp. and fieitconostoc spp., acetic acid bacteria
such as Acetobrtcter zyy. and Enterobacteriaceae. They ascribe their Jaresence to a lack
of iisepsis during the kefir manufacturing process. However, other authors consider
/>ncfococcus spp. and Leiiconosloc s}i}i. as normal flora of kefir grains (Pidoux, 1089 ; i-
1 osono ct nf. 1590 ; Rossi & Gobbetti, 1991).
The main function of the microorganisms constituting the grains is the production
of lactic acid by fei’inentation from lactose, as well as the production of peroxides,
antibiotics indd biictet icide, which allow the kefir milk to delay considerably the
development of ‹legrading and pathogenic microorganisms. There is also evidence
that l‹efir can restore normal intestinal flora after upsets caused by disease or
treatment u itli antibiotics (Rehin, 1983).
On account of the health and nutritional value of this product, the microbiological
quality of kefir grains in Galicia has been investigated with regard to their
distribution and specific diversity . The most frequently isolated species considered
constituents of the grain were chai icterized and distinguished from the con-
taminatiig bacteriiil species.
264 L. AN GULO AND OTHERS

MATERIAL AN£i FI.I'THO US

Eight kefir grains, in a fresh condition, were obtained directly from individual
dairies in various parts of the Galician region. These grains ( > .5 yeiirs old) were
supported on ra» or pasteurized cows’ milk according to the sampled zone. In our
laboi’ator3, the grains » ere propagated b3 twice- or tlirice-week ly tixinsfer into
pasteurized cows’ milk (1 1) and incubated at room teinpei-ature. f?ach of the sainJales
was cut so that a portion of the grain’s interior coulcl be obtainecl. ’i’1iese pieces mere
thoroughly washed with sterile water and ground in 1 % (w/v) sterile saline (10 ml).
Portions (0-1 ml) of a known dilution were plated in duplicate on the dift’erent culture
media.

Isolation altd identification of |jeasts

The isolation of yeasts was carried out b3 surface spreading on plates of malt
ex ti ref agar and yeast extract-malt extract agar (Kreger-van Rij , 1i)84). After
incubation iit 28 °C for 3-6 d colonies of different iroi’pliology were obtained. ’I’he
methods of’ ltreger-van Rij (1984) and Barnett ct a/. (1900) were used for their
iden tificiition .

Isol.ation and identificotion of lactic acid bacteria

For isolation of I AB belonging to the Loctobocillus, Leticonosioc and I’e‹l.iococcus
genern, 0-1 ml portions were plated in AIRS broth (Difco, Detl oit, MI 48232, L SA)
supplemented u itli agar (2 % w/v) and Rogosa agar (Difco). Cyclolieximide (0 05 %
iv/v) mas added to inhibit the yeast gro» th. The plates » ere incubated at 30 °(? for 7
—12 d in ‹aerobic and anaerobic (10 % CO,) atmospheres. For the isolation of lactic
streptococci. azide agar medium (Difco) mas used aerobically. ’lie colonies mere
provisionally considered LAB on the basis of their cell ular rnorpliology, ( ram -
positive and catalase-negative reactions. The criteria applied in the taxonomic
characterization mere those described by Garvie (1986 c, h), Harclie (1i)86) and
I(and ler ñ \Veiss (1986).

Isolrttion aitd ideittiJcntion of non-LAB confniiiiitoting hacieriu

For the isolation of species belonging to the bacterial groii ps, the irediii used (all
from l)ifco) were tr3 ptic soy agar (fiactffus), MRS » ith 2 'to (iv/v) agar (A retobacter
and Microcotcus) and MacConkey agar (Enterobacteriaceae). ')’he plates » ere
incu hated ior 2—10 d at 28 °C, or at 37 °C in the case of the l£nterobacteriaceae.
Isoliited colonies were identified exclusively at the genus level, following the
classification schemes proposed by De Ley eJ at. (1984), Claus & Eerl‹eley (li)86) and
I(ociir (t986).
ItFSULTS AND DISGUSS ION

From the interior of the kefir grains 49 yeast stiains corresponding to five genei'a
mere isoliited, in addition to 46 LAB strains representing four genera, anal 18
bacterial strains considered contaminating, representing five genei’a. 'i’he distribution
of species in each grain is shown in Table 1.

"I'or1ilasyora deffiruec£ii and 6nccfiaroiiiyces cerevisioe were isoliited from most

grains* ( rF able 1), and were 13.3 and 10‘6 % of the total of yeast sJiecies isolated.
A relevant characteristic of these yeasts was their inabilit,y to ferinelit lactose,
which
 Nicroflora off ke/r grains 265

’I’iible 1 . iYiiiither oJ representative colon,ie8 of ljeast a,nd bacterial syecie8 isolates! from

1 2 :3 4 5 (i 7 8

•fi‹irrIiai’om¡jres cereui,sinc
‹Arr. /r// i.spor ti,s
Cn uili‹l‹i krfyr
I’m n‹l. holutii

GlyniJi'erciiiiyre.s lnrlis 2 — — 1

Lnctol›ririIlu,s Grrt›ia :J 2

/>t/. rn.sri s\I hs}J. loleru ii,s 1

/ ocfororr tt,s fricti,s su bs)i. fnctis

l earoito.sfoc s}ip. I I
— 1 I
'‘if rr ylororr us s‹ilira ri as sti bs}a. IheritioJ›li il its
t’‹anttirn iiiiints
lord iororr us epp.
.1/irrorocc« s s i i
1 1 1
1 I
N refofiricf er spp.

made them dependent on lactic bacteria capable of hydrolysing this disaccharide.

’l’he predoinirnince of lactose-negative yeasts in the grains studied was consistent
with the studies carl-ied out by Van Uden & Assis-Lopez (1955), La Riviére (1960)
iind P•osi ( 1.078 o). ’J“u'o l‹ictose-}›ositive yeast species [C'andida kefir and Hlulyvero-
iit’iyces lactis) were isolated from four grains, with the peculiarity of always appearing
u'itli other lactose-negative yeasts.

f>ocft‹: actor h‹i‹:terirt

Nine lioinofermentati ve and heterofermentative lactobacilli species were identi-
ñed. \V ithin the homoferrnentative lactobacilli group the following were isolated :
1>oclob‹i,ci,llits ‹,’a set su bsp. tolerant, Lb. casei su bsp. pseudopl‹intaritm, Lb. laser ciibsp. Y!
tGI1IIIOS1tS, lb. OGJJO' liiiits and fi6. ya,sseri , in the lieterofermentative lactobacilli gi’oti}i
Lh. breuis, Uh. virid.escens, Lb. kefi.r and Qtr. Jernientuni were isolated (Table I ). Store
lieteroferinentative lactobacilli were isolated than homofermentative strains (74‘3 '%
u. 25-7 %). LG. breUts was isolated from six grains and represented 15 '% of the total of flora
isolated. Isa Riviére ct o/. (1967) and Kooiman (1968) found a predorn iniulce of
Lb. bre is over the rest of the LAB in kefir grains, while Kandler &
)kiiniitli (1583) found a predominance of th. kefir, which we isolated from only one
gi tin.
Lar/ococcits !ocIis siibsJi. lactis was isolated from most kefir grains, with an
isolation percentage of 6-2 %. Slreytococcus salt ‹iriu8 subsp. thermophilu8 and
1>eiicoiiosIoc spp. appeared in onl3 th ree kefir grains. Brooker (1976), Olson &
266 L . AN€IULO AND OTHERS

Table 2. Isolation percentage of niicroflora present in the eight k,efir grains analysed
Grain no.

1 2 3 4 .5 5 7 8
Yeasts
l>i*ctic ticid bacteria
(iontnrninarits

Alocquot (1985), ))uitscliaever ct al. (1987), Pidoux (1989), Hosoiio et at. (1990) and
Rossi & Gobbetti (1991) also noted the presence of these LAJ3 as integral floi’a of kefir
grains.

Contuniinunls
Different bacterial species belonging to five genera were isolated from the kefir
grains analysed (’i’able 1). The presence of these species would depend on the different
microbiological qualities of the milks used and/or lack of asepsis during the
manufacture of the product. ’1he isolation of species of the Bacill,us, Vicrocorcus or
Pediococcus genera from u ntreated or p‹isteurixed milk has been reported by various
authors (V'est1ioff & Doughert3, 1981 ; Fernandez del Pozo ef n/. 1088 ; Angulo ct of.
1989 ; Mahari â Gashe, 1990). Another source of contamination of kefir grnins is the
lack of h3 giene during treatment, when Acelobacter and Enterobacteriaceae species
can be introduced (Rosi, 1978 b ; Rosi c4 Rossi, 1978).
’Pable 2 shows the isolation percentages of‘ each category of species (yeiists, I AU
and contaminants) in each grain. ’lhese results do not refer to cellular densities, but
nevertheless give an estimate of the relatix•e diversity of each category.
’lhere was a predominance of heterofei inentative LAE in the grains studied.
These LA)3, in addition to brealcing down lactose to lactic acid, generate seconcl ary
metabolites which affect the kefir quality. There was a proportion of containin‹iting
bacteria which can also ti-ansfer trrldesirable organoleptic characteristics to the kefir
milk (Olson & Mocquot, 1985 ; Duitschaever ct a/. 1988).
’i'he results obtained indicate differences in microflora among the grains studied.
These differences could be explained mainly b3* the different origins of the l‹efir
grains.
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