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Research ArticleBiodiversity and 120574-Aminobutyric Acid Production byLactic Acid Bacteria Isolated from Traditional Alpine RawCowrsquos Milk Cheeses

Elena Franciosi1 Ilaria Carafa1 Tiziana Nardin2 Silvia Schiavon2 Elisa Poznanski13

Agostino Cavazza1 Roberto Larcher2 and Kieran M Tuohy1

1 Research and Innovation Centre Department of Food Quality and Nutrition Fondazione Edmund Mach (FEM) Via E Mach 138010 San Michele allrsquoAdige Italy

2 Technology Transfer Centre Fondazione Edmund Mach (FEM) Via E Mach 1 38010 San Michele allrsquoAdige Italy3 Environmental Province Agency Office 299 Laboratorio Biologico Via Sottomonte 2 39055 Laives Italy

Correspondence should be addressed to Elena Franciosi elenafranciosifmachit

Received 17 June 2014 Revised 4 September 2014 Accepted 2 October 2014

Academic Editor Marıa Fernandez

Copyright copy 2015 Elena Franciosi et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

ldquoNostrano-cheesesrdquo are traditional alpine cheesesmade from raw cowrsquosmilk in Trentino-AltoAdige ItalyThis study identified lacticacid bacteria (LAB) developing during maturation of ldquoNostrano-cheesesrdquo and evaluated their potential to produce 120574-aminobutyricacid (GABA) an immunologically active compound and neurotransmitter Cheese samples were collected on six cheese-makingdays in three dairy factories located in different areas of Trentino and at different stages of cheese ripening (24 h 15 days and 12 3 6 and 8 months) A total of 1059 LAB isolates were screened using Random Amplified Polymorphic DNA-PCR (RAPD-PCR) and differentiated into 583 clusters LAB strains from dominant clusters (119899 = 97) were genetically identified to specieslevel by partial 16S rRNA gene sequencing LAB species most frequently isolated were Lactobacillus paracasei Streptococcusthermophilus and Leuconostoc mesenteroides The 97 dominant clusters were also characterized for their ability in producingGABA by high-performance liquid chromatography (HPLC) About 71 of the dominant bacteria clusters evolving duringcheeses ripening were able to produce GABA Most GABA producers were Lactobacillus paracasei but other GABA producingspecies included Lactococcus lactis Lactobacillus plantarum Lactobacillus rhamnosus Pediococcus pentosaceus and Streptococcusthermophilus No Enterococcus faecalis or Sc macedonicus isolates produced GABA The isolate producing the highest amount ofGABA (800 plusmn 27mgkg) was a Sc thermophilus

1 Introduction

Traditional alpine raw milk cheeses are commonly producedin alpine regions including the province of Trentino inNorth-Eastern Italy Here they are called ldquoNostrano-cheesesrdquo andare semicooked cheese made by mixing approximately in 1 1ratio the raw cowrsquos milk from two different milking Thefirst milking is carried to dairy factory the evening beforethe cheese-making and is stored in large shallow tank for9ndash11 hours where a spontaneous creaming occurs After thisovernight stage the partially skimmed milk under the creamin the tank ismanually drained from the cream fat and placedin the cheese-making vat The whole milk from the morning

milking the second milking is then added to the skimmedmilk No commercial lactic starters are added and the naturalmilk microbiota obtained from the overnight skimmed milkinitiates the acidification process The vat milk is coagulatedby commercial rennet and after the manual cutting the curdis cooked at about 48∘C After moulding and salting theripening is held at about 18∘C for 3 to 8 months

The milk for ldquoNostrano-cheesesrdquo typically comes fromHolstein Friesian andor Brown Swiss cattle breeds which arefed differently during the year The cows are typically fed onhay during the cold season in the valleys and from late Juneto middle September (summer season) are grazed on highmountain alpine pasture

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 625740 11 pageshttpdxdoiorg1011552015625740

2 BioMed Research International

It has been reported that the use of commercial starters inrawmilk cheesesmaymodify the characteristics of the cheesemicrobiota in particular lowering the microbial biodiversity[1] and it is also well known that mainly LAB microbiotadeveloping during ripening influences the typical organolep-tic characteristics of the cheese [2] Thus LAB represent afundamental process factor for the final attributes and qualityof artisan dairy products such as alpine cheeses Severalstudies have focused on the genotypic and technologicalcharacterization of LAB isolated from different traditionallyfermented cheeses [3ndash6] but little work has so far been doneon ldquoNostrano-cheesesrdquo

In addition to the technological relevance of LAB incheese there is currently much research and industry interestin the potential biological activity of dairy LAB either foruse as probiotics in their own wright or as bioactive agentscapable of modulating the health functionality of cheese andother dairy products [7] Rawmilk cheeses have already beenidentified as a useful source ofmicrobial biodiversity and newLAB strains with health promoting properties [8]

Since caseins are rich in glutamate which is released byproteolytic action the decarboxylation of this amino acidinto 120574-aminobutyric acid (GABA) can have an importanteffect on the formation of eyes in cheese [9] Besidesits technological effect in cheese GABA has several well-characterized physiological functions in mammals includingneurotransmission induction of hypotension diuretic andtranquilizer effects and stimulation of immune cells [10ndash12] Some studies have reported also that GABA derivedfrom the gut may be a neuroactive molecule within thegut-brain axis [13] which is a complex communicationhighway linking the gut environment with both the centraland peripheral nervous systems Strains of Lb buchneri [14]Lb brevis Lb paracasei and Lb plantarum [15] isolatedfrom traditional cheeses have been shown to produce GABAGABA-producing LABhave not been isolated and extensivelycharacterised from traditional alpine cheeses produced inTrento though the presence of GABA in these cheeses hasbeen confirmed and its concentration at the end of ripeningreported at between 120 and 1739mgkg [16] which is highcompared to other Italian cheese varieties (typically 0260to 391mgkg) [15] Therefore the objective of this study wasto analyze the diversity and the successional development ofLAB in traditional ldquoNostrano-cheesesrdquo from the Trento alpsduring cold and summer seasons and to extensively screenand identify GABA-producing LAB isolates

2 Materials and Methods

21 Cheese Factories and Milk Sampling Cheeses were sam-pled in three dairy factories (called B C andD according to aprevious paper [17]) located throughout the Trentino regionand producing traditional alpine cheeses called ldquoNostrano-cheesesrdquo Each factory collected milk from farms within a15 km radius

Two cheese batches from each dairy factory one in Febru-ary and the other in July were sampled making a total of sixbatches subjected to microbiological analyses All factories

processed milk obtained from stabled cows fed with hay dur-ing the ldquocold seasonrdquo from October to May and high moun-tain pasture fed cattle in the summer season from June toSeptember For each of the six batches at least five cheesesamples at different stages of ripening were collected (24hours 15 days 1 month 2 months and 3 months and for fivebatches also 6 and 8 months) making a total of 40 cheesesamples per factory

22 Enumeration and Isolation of Microorganisms Cheesesamples (25 g) were homogenized (2min at 260 rpm) usinga stomacher (laboratory blender stomacher 400 SewardLondon UK) in 225 g peptone water (01 mycologicalpeptone (Oxoid Basingstoke UK)) and serially dilutedDilutions were plated and incubated as follows onto MRSagar acidified to pH55with 5molL lactic acid anaerobicallyfor 2 days at 30∘C and 45∘C for mesophilic and thermophilicrod-shaped LAB respectively onto MRS agar added withvancomycin (8120583gmL Sigma-Aldrich Saint Louis MIUS)[18] and acidified to pH 55 with 5molL lactic acid anaerobi-cally for 72 h at 30∘C for mesophilic heterofermentative rod-shaped LAB onto M17 agar for 2 days aerobically at 30∘Cand anaerobically at 45∘C for mesophilic and thermophiliccoccoid LAB respectively onto KAA aerobically for twodays at 37∘C for enterococci onto PCA added with 10 gLskimmed milk aerobically for 24 h incubated at 30∘C fortotal bacterial count (TBC)All culturemediawere purchasedfrom Oxoid

At least three colonies were picked from each count-able plate Gram-positive colonies (as determined by KOHmethod [19]) and negative to the catalase test (as determinedby transferring fresh colonies from agar medium to a glassslide and adding 5 H

2O2) were isolated Cell morphology

was determined bymicroscopic observation Each isolate waspurified by subsequent culturing onto M17 or MRS and purecultures were stored at minus80∘C in glycerol (20 vv) stocks

23 DNA Extraction and RAPD-PCR DNA was extractedfrom overnight broth cultures of isolated strains Cells werecentrifuged at 10000timesg for 5min and the pellets werewashed twice in sterile distilled water and suspended in1mL of distilled water Cell lysis was achieved using theInstageneMatrix (Bio-Rad Hercules CAUSA) following themanufacturerrsquos instruction

RAPD-PCR was carried out in a total volume of 25120583Lusing primer PC1 [20] Cluster analysis of DNA patternswas carried out using GelCompar II-BioNumerics software(package version 60 Applied Maths Belgium) exploitingthe unweighted pair group method arithmetic averages(UPGMA) Similarity of PCR fingerprinting profiles wascalculated based on Pearson product-moment correlationcoefficient The threshold breakpoint value was fixed to 80isolates with similarity coefficient higher than 80 wereclassified into the same cluster according to Gatti et al [21]

24 Genotypic Identification of LAB One isolate representa-tive of each LAB cluster was genotypically identified by 16SrRNAgene analysis All isolates fromM17 45∘Cwere tested by

BioMed Research International 3

Table 1 Bacterial counts from cheese (119899 = 6 for each time ripening) sampled at 24 h 15 days 1 month 2 months 3 months 6 months and 8months

Ripening time Agar media (log cfug)PCA MRS 45 MRS 30 MRS VAN M17 45 M17 30 KAA

24 h 85 plusmn 08 52 plusmn 06 53 plusmn 06 46 plusmn 14 85 plusmn 06 66 plusmn 06 56 plusmn 1415 d 90 plusmn 05 51 plusmn 07 81 plusmn 08 77 plusmn 06 79 plusmn 12 78 plusmn 06 62 plusmn 101 month 88 plusmn 04 62 plusmn 10 83 plusmn 05 81 plusmn 05 78 plusmn 09 78 plusmn 08 57 plusmn 122 months 87 plusmn 03 63 plusmn 08 80 plusmn 04 80 plusmn 04 72 plusmn 07 81 plusmn 03 57 plusmn 143 months 84 plusmn 06 57 plusmn 07 78 plusmn 07 75 plusmn 07 71 plusmn 09 67 plusmn 16 57 plusmn 066 monthsa 77 plusmn 06 56 plusmn 15 70 plusmn 04 75 plusmn 09 69 plusmn 10 59 plusmn 14 57 plusmn 068 monthsa 69 plusmn 07 52 plusmn 10 65 plusmn 09 65 plusmn 06 60 plusmn 11 46 plusmn 15 46 plusmn 10a119899 = 5

Sc thermophilus species specific PCR according to Lick et al[22] isolates from M17 at 30∘C by Lc lactis lactiscremorisspecies specific PCR according to Delorme et al [23] and allthe other isolates fromMRS at 30∘Cby Lb casei Lb paracaseiand Lb rhamnosus species specific PCR with the primers Y2Casei Para and Rham described byWard and Timmins [24]If the species specific PCR gave a negative result identifi-cation was carried out by using 16S rRNA gene sequencingThe sequence analysis of a 16S rRNA fragment gene was per-formed using 27f (51015840-GAGAGTTTGATCCTGGCTCAG-31015840)and 1495r (51015840-CTACGGCTACCTTGTTACGA-31015840) primersdesigned by Grifoni et al [25]

The obtained PCR products (ca 30 ng) were purified withExo-SAP-IT kit (USB Co Cleveland OH) and sequencedthrough the BigDye Terminator v11 cycle sequencing kit(Applied Biosystems Foster City CA) as reported by themanufacturer in an ABI PRISM 3100 sequencer (AppliedBiosystems) Species were assigned after comparison of theobtained sequences by BLAST alignment (httpwwwncbinlmnihgovBLAST)

All the amplicons were analyzed by electrophoresis on25 (wv) agarose gel (Gibco BRL Cergy Pontoise France)at 100V for 90 minutes in 1X TAE buffer and were revealedby staining with ethidium bromide (05120583gL) All amplifica-tions were performed with a T100 Thermal Cycler (Bio-RadLaboratories)

25 120574-Aminobutyric Acid (GABA) Production and Quan-tification Glutamate decarboxylase (GAD) activity of LABisolates and the production of GABA were checked usingthe method of Nomura et al [26] with some modificationscultures were centrifuged (9000 rpm for 15min at 4∘C)washed twice with sterile PBS and suspended in sterile 085NaCl solution in order to achieve the 119860

620 nm value of 25Afterward 100120583L of cell suspension was mixed with 900120583Lof 50mM sodium acetate buffer (pH 47) containing 70mML-glutamate and 01mM pyridoxal phosphate The reactionmixture was incubated for 24 h at the same temperature ofisolation (30∘C for mesophilic and 45∘C for thermophilicisolates) and filtered through a 022120583m pore size filter(Minisart Sartorius Stedim Biotech Goettingen Germany)The sample diluted 10 times with sodium tetraborate 01M(pH adjusted to 105) and added to glycine as internal

standard to a final concentration of 10mgL was stored atminus20∘C before the analysis L-Glutamic acid glycine andGABA were quantified as o-phthalaldehyde (OPA) adductsmodifying themethod proposed by Lehtonen [27] in order tonotably reduce the time of separation to only 27 minutes butwithout worsening selectivity and accuracyThis was possiblein the light of the specifically designed and perfectly knownmatrix

The measures were performed using an UHPLC Ulti-mate 3000 (Thermo Fisher Scientific Waltham MA USA)equipped with a fluorescence detector (Ex = 336 nm Em =445 nm) Separation was carried out with sodium acetate005M (pH adjusted to 75 eluent A) and methanol (eluentB) using a column Chromolith Performance RP-18e (100 times46mm Merck Darmstadt Germany) with Guard CartridgeChromolith RP-18e (10 times 46mm Merck) at 40∘C The flowrate was set at 2mLmin The analytical gradient for eluentB was as follows 40 for 30 sec 25 for 90 sec 100 for30 sec and 60 for 15 sec The sample (10 120583L) kept at 10∘C bythe autosampler was automatically introduced into the loopadded with 10 120583L derivatising solution mixed for 1min andinjected The derivatising mix was 45 gL of OPA (Sigma-Aldrich) in sodium tetraborate 01M corrected to pH 10510 methanol and 2 2-mercaptoethanol (Sigma-Aldrich)The detection limit for GABA was estimated at 0025mgL (3times the standard deviation of the GABA contentsmeasuredrepeating 10 times the analysis of a sample at unquantifiablecontent)

3 Results

31 Microbial Cell Counts The microbial populations ofNostrano-cheese samples were estimated on different selec-tive media (Table 1) The total bacterial counts were in therange of 8-9 log cfug from 24 h to 3 months of ripeningand decreased after 6 and 8 months by 1 order of magnitudeThe thermophilic cocci reached the highest counts after24 h of ripening (mean values of 85 log cfug) mesophiliccocci reached the highest counts after 2mo of ripening(mean values of 81 log cfug) Enterococci were neverdominant and reached their highest count after 15 days ofripening (mean values of 62 log cfug)The lactobacilli group(counts onto MRS at 45 and 30∘C) was higher after 1moof ripening The growth dynamic of the different microbial


groups was different (Table 1) thermophilic cocci counts(onto M17 45∘C) were dominant in the first 24 hours after15 days to 3 months of ripening mesophilic cocci (onto M1730∘C) and lactobacilli counts (onto MRS and MRS VAN30∘C) started to increase and were dominant together withthermophilic cocci finally at the end of ripening (6 and8 months) mesophilic lactobacilli and thermophilic coccimaintained the dominance within the alpine cheeses micro-biota

On average three colonies for each colony morphologywere isolated in pure culture from eachmedium For summerseason at 6 and 8 months only two types of cheese wereavailable and sampled in dairy factories C and D A totalof 1105 isolates were collected From the total number ofisolates 46 were discarded from further analysis as nonlacticacid bacteria (they were found positive to catalase andnegative to KOH tests) The remaining 1059 strains werecharacterised for cell morphology 677 were cocci and 382were rods (Table 2)

32 Molecular Clustering of LAB Isolates and Species Identi-fication All putative LAB isolates were analyzed by RAPD-PCR as a first grouping into clusters The isolates from thesame kind of cheese showing a RAPD similarity coefficient ofat least 80 were considered as belonging to a single clusterThe RAPD-PCR analysis grouped 1059 LAB into 583 clusterswith 80 similarity index (results not shown) From theseclusters 276 isolateswere selected for further analysis becausethey belonged to the dominant microbial populations asenumerated by plate counts on MRS MRS VAN at 30∘CM17 at 30 and 45∘C The RAPD-PCR analysis of these276 dominant isolates discriminated 97 different clustersdefined at a minimum similarity level of 80 (Figure 1)The 97 clusters were designated using a progressive numberfollowed by the letters B C or D to indicate the dairyof origin of the clustered isolates (Figure 1 clusters 1D to97C)

Species identification was performed by species specificPCRs or partial 16S rRNA gene sequencing Table 3 showsthe results of bacteria identification for each ripening timeThe highest diversity within a single species was found forLb paracasei and Sc thermophilus with 35 and 23 differentgenotypes respectively

Lb paracasei was the dominant species (72 isolated onMRS and 16 onM17 agar plates) followed by Sc thermophilus(50 isolated on M17 agar) and Ln mesenteroides (27 isolatedon MRS and 18 on M17 agar) A different dominant speciessuccessional development was observed in cheeses at differ-ent ripening time Sc thermophilus was always dominant inthe first 24 hours and one of the codominant species for upto 2 months of ripening Lc lactis species was also foundcodominant in cheese at 24 h ripening with Sc thermophi-lus Streptococci and enterococci species were recorded inabundance in the first three months but largely disappearedafter six months and at the end of ripening (Table 3)We did not find difference in species distribution betweencheeses sampled in February and July The same specieswere recorded both in cold and in summer season (data notshown)

After the genotypic characterization 97 strains onerepresentative of each dominant cluster were processed forthe detection of GABA production

33 GABA Production Sixty-eight isolates out of the 97different clusters synthesized GABA (GABA amount gt025mgkg) after 24 h of incubation at 30 or 45∘C in presenceof glutamic acid (Table 4)They grouped 195 of the dominantisolates (71 of the tot) and in particular three (1 Lb para-casei 1 Lb rhamnosus and 1 Sc thermophilus) were able toproduce GABA concentrations higher than 10mgL (Table 4lines in bold)The Sc thermophilus cluster 84C showed thehighest glutamate decarboxylase activity generating a meanvalue of 80mgL of GABA (Table 4 first line) No Ec Faecalisor Sc macedonicus isolate was able to produce amount ofGABA higher than 025mgkg

4 Discussion

In Italy the province of Trento has a long dairy historywith various dairy biotechnological traditions arising fromthe geographical challenges of transport and communica-tion between different alpine valleys and a diverse culturalheritage A wide range of cheeses coexist each with theirown specific biotechnological processes organoleptic char-acteristics and history A previous review has discussed theimportance of preserving this type of traditional artisancheese usually made from raw cowrsquos milk because of theirhigh microbial biodiversity and in particular high speciesrichness of ldquowildrdquo LAB with diverse metabolic activities andof great potential as dairy starters or even probiotic agents[8] Previous work has shown that the ldquoNostrano-cheesesrdquocontain high concentrations of GABA compared to otherItalian cheeses [16] and it is known that LAB is responsiblefor producing GABA in cheese [15] We therefore selectedTrento ldquoNostrano-cheesesrdquo for the screening and isolation ofGABA-producing LAB

The successional development of the lactic microbiota ofsixldquoNostrano-cheesesrdquo from 24 hours to 8 months of ripeningwas characterised 276 isolates belonging to dominant lacticmicrobiota were grouped into 97 clusters identified to thespecies level and screened for their GABA production Themilk used to produce these cheeses was the subject of aprevious report [17] but in summary the microbiologicalcharacterization was in agreement with microbial countsreported for other traditional Italian cow raw milk cheeses[6 28 29]M17 andMRSwere not perfectly selective in agreewith previous works [6 30] in fact some nontarget isolationswere recorded for example 2 Lc lactis isolates amongst 13were found on MRS agar plates and about 14 of all rod-shaped isolates were isolated on M17 agar plates

As commonly found in many raw milk cheeses [28ndash30] themicrobial composition of the ldquoNostrano-cheesesrdquo wasdominated by LAB Lb paracasei was the most abundantspecies (319 of the isolates) followed by Sc thermophilusand Ln mesenteroides (181 and 163 resp) These specieswere amongst the dominant microbiota at all productionstages in particular Sc thermophilus dominated after 24 h


34C

64C

9D

86C13D

39C57D

73C-D80B81B

91B

68B

40C

95B

72D

59B58C

26D

3B

31C

56D55C

67B

71B

63C

12D

27B28B

65B66B

94B

43B

49B

8C

32C33C

48B

15C14C

18C

62B

53D

54C

11C10C

87C

88C

47C

38C37C

77B

82C83C

21B-D30D

29D

52B-C

24D25D

78C

79B

76D

61C60C-D

19D20B-C

46C-D

17C

16D

69B70B

97C

96C

50D51B

4B

5B

89B

90B

84D85D

6C7C

36D 35C41D42B-C

74C-D75C

2D1D

92B93B

22B23B

45C-D

44C-D

Lb paracasei and Lb caseiSc thermophilusLn mesenteroidesEc faecalisLc lactisLb curvatusLb plantarum

Pc pentosaceusLb acidipiscisSc macedonicusLb coryniformis sspLb rhamnosusLb delbrueckii

Figure 1 Unrooted dendrogram of the 276 dominant isolates obtained from RAPD-PCR patterns using the Pearson product momentcorrelation coefficient (119903) and the unweighted pair group algorithm with arithmetic averages (UPGMA) Each circle-pie is a cluster andthe number of slices represents the number of isolates for each cluster GABA producer clusters are indicated by a yellow circle Each color isa different species

until 2 months of ripening while Lb paracasei and Lnmesenteroides reached their highest levels in the cheese after15 days and remained at high levels until 3 months ofripening with a similar trend observed in microbial countsonMRSwhich started to decrease after 6months of ripeningprobably the result of microbial autolysis [31 32] Another11 different LAB species were found in the cheese samplesbut none at a relative abundance higher than 5 All speciesidentified were previously recorded and very common in thedairy environment [4 6 28ndash30] with the exception of Lb

acidipiscis which is a species described by Tanasupawat etal [33] and isolated from fermented fish and has also beenisolated more recently from traditional Greek cheeses [34]

RAPD analysis displayed a great genetic diversityamongst the isolates In fact about 33 of the RADP clusterswere singletons (one cluster for one isolate) This geneticbiodiversity may reflect a real picture of the high speciesrichness amongst the isolates collected from the cheesesbut could also be consequence of the large number ofstrains analysed in this study A similar result was found


Table 2 Number of putative LAB strains (coccirods) isolated from each plate at different sampling times in traditional alpine cheeses (119899 = 6)

Ripening time Agar media (coccirods) TOT number of LABisolates (coccirods)MRS 45 MRS 30 MRS VAN M17 45 M17 30 KAA

24 h 12(75)

19(154)

8(80)

18(180)

20(182) 22 (211) 99

(8811)

15 d 8(80) 38 (1028) 15

(114)18

(171)31

(247)27

(225)137

(8255)

1 month 19(109)

43(1528)

42(1923)

31(283)

34(286) 21 (201) 190

(12070)

2 months 28(1612)

54(1719)

49(2039)

36(351)

46(3313)

22(193)

235(14095)

3 months 18(135)

48(2127)

46(2422)

25(241)

23(221)

24(177)

184(12163)

6 monthsa 8(44) 30 (1812) 25

(817)16

(133)22

(220)12

(93)113

(7439)

8 monthsa 15(510)

20(515)

16(412)

25(196)

16(151)

9(54)

101(4848)

TOT 108(6345)

252(101151)

201(84117)

169(15415)

192(16230)

137(11324)

1059(677382)

a119899 = 5

in a previous work where 206 isolates from spontaneouslyfermented cheeses were analysed by RAPD PCR [30]

We compared all the clusters recovered from ldquoNostrano-cheesesrdquo with those found in the correspondingmilk samplesand reported in a previous report [17] and no milk RAPDpattern was found amongst the 586 cheese clusters Thismay be because the fermentation is not spontaneous butdriven by a starter culture from the overnight skimmed milkthat even if natural and not commercial may inhibit milkmicrobiota growth and development It is worth highlightingthat some isolates from different dairies grouped within thesame cluster The 9 species occurring in different dairieswere Lb paracasei Lb rhamnosus and Ln mesenteroidesThese few coincident clusters occurred often in different dairyenvironments and might represent part of an endemic geo-centric cheese microbiota not necessarily coming frommilkbut adapted to the cheese-making practice ripening andlocal microclimate and environmental conditions specific tothe Trento alps On the other hand 34 of the 97 clusters wereRAPD-PCR singletons and some species like Lb coryniformisssp torquens Lb acidipiscis Lb curvatus and Lb delbrueckiiwere peculiar only for one of the three dairy factories Theseaspects suggest that each manufacturing facility may also becharacterized by a unique microbial population

Considering the recent interest in the gut-brain axisthe potential role of neurotransmitters like GABA in theperiphery and the immunological potential of systemicGABA we screened the 97 dominant clusters for GABAproducing strains [35] A total of 68 GABA producing strainswere identified Previous studies by Siragusa et al [15] andmore recently by Diana et al [36] found that sheep milkcheeses contained higher levels of GABA than cowrsquos milkcheeses and consequently had higher numbers of GABA-producing LAB The raw cowrsquos milk cheeses subject of thiscurrent study showed higher amounts of GABA at the endof ripening [16] and a higher percent of GABA producer

strains (71) than these two previous studies where GABAproducing strains were less than 14 This difference may bedue to the peculiar traditional environment of production ofthese Trento cheeses However it may also be the result of thecheese production times sampled We screened the isolatesstarting at 24 h and followed the cheese LABmicrobiota untilthe end of ripening It is probable that microbial GABAproduction follows the same trend as the bacterial growthwith higher number of GABA producing strains in the first3 months followed by a rapid decrease in LAB numbers

Amongst the 68 positive strains 13 GABA producingstrains gave more than 4mgkg and belonged mainly to Lbparacasei species but also to Lc lactis Lb plantarum Pcpentosaceus Lb rhamnosus and Sc thermophilus The abilityto produce GABA has been reported in various LAB inparticularLactobacillus sp isolated from fermented food [37]Lc lactis and Sc thermophilus were also found to produceGABA in different Italian cheeses screened by Siragusa etal [15] and Pc pentosaceus was isolated as high GABAproducing strain from aThai fermented meat [38] From ourscreening no Ec faecalis or Sc macedonicus strain was able toproduceGABA (less than 025mgkg) and to our knowledgethese species have never previously been identified as GABAproducers

GABA is a desired bioactive compound because of itsphysiological functions such as neurotransmission inductionof hypotension diuretic and tranquilizer effects and stim-ulation of immune cells [10ndash12] For these beneficial effectsGABA has been introduced in the diet as an oral supplementthe Japanese government defines the foods enriched withGABA as ldquofoods for specified health userdquo [3] Fermentedmilk enriched in GABA produced by lactobacilli may havecommercial potential as a health-oriented dairy product

Siragusa et al [15] observed that Lb paracasei Lbdelbrueckii subsp bulgaricus Lc lactis Lb plantarum and Lbbrevis strains isolated from different Italian cheese varieties


Table3Num

bero

fisolates

andspeciesfou

ndateach

ofthes

amplingtim

eintradition

alalpine

cheeses(119899=6)

Ripening

time

Lb

paracasei

Sc

thermophilus

Ln

mesenteroides

Ec

faecalis

Lc

lactis

Pc

pentosaceus

Lb

acidipiscis

Sc

macedonicu

sLb

casei

Lb

coryniform

isLb

rham

nosus

Lb

curvatus

Lb

plantarum

Lb

delbrueckii

Total

dominant

isolates

24h

mdash19

mdashmdash

6mdash

mdash2

1mdash

mdashmdash

mdash28

15d

208

31

11

mdashmdash

4mdash

2mdash

3mdash

431m

onth

2010

123

22

mdashmdash

mdashmdash

25

mdashmdash

562mon

ths

1410

138

mdash2

mdash4

32

21

mdash1

603mon

ths

153

131

mdash5

mdash5

23

mdashmdash

mdashmdash

476 mon

thsa

12mdash

1mdash

12

6mdash

mdashmdash

mdashmdash

mdashmdash

22

8 mon

thsa

7mdash

3mdash

3mdash

5mdash

mdash2

mdashmdash

mdashmdash

20

TOT

8850

4513

1312

1111

107

66

31

276

a 119899=5


Table 4 GABA production (Mean plusmn sd) of the dominant clusters in sodium acetate buffer containing 70mM L-glutamate after incubationfor 24 h at the same temperature of the isolation medium Clusters are ordered from the highest to lowest GABA producer

Cluster Species Isolate number Ripening time Isolation medium and temperature GABA production (mgkg)84C Sc thermophilus 3 24 h 1-2months M17 45 800 plusmn 2715C Lb paracasei 4 3ndash8 months MRS 30 148 plusmn 5321D-B Lb rhamnosus 6 24 h 1-2months MRS (30 VAN) 113 plusmn 07224D Lc lactis cremoris 2 24 h 6months M17 30 90 plusmn 02162B Pc pentosaceus 1 1month MRS 30 67 plusmn 06180B Lb plantarum 2 15 d MRS 30 52 plusmn 05659B Lb paracasei 1 15 d MRS VAN 49 plusmn 02070B Lb paracasei 1 15 d MRS VAN 46 plusmn 02122B Lb paracasei 3 15 d MRS (30 VAN) 42 plusmn 04149B Lb paracasei 1 1month MRS 30 40 plusmn 0854C Lb paracasei 1 3months MRS 30 40 plusmn 06973D-C Lb paracasei 3 1ndash6months M17 30 MRS 30 40 plusmn 02920B-C Lb paracasei 3 15 d 3months MRS 30 40 plusmn 01866B Lb paracasei 1 15 d MRS 30 40 plusmn 01877D Lb paracasei 18 1-2ndash6months M17 30 MRS (30ndash45 VAN) 37 plusmn 07168B Lb casei 3 2months MRS 30 36 plusmn 03225D Lc lactis lactis 1 15 d M17 30 28 plusmn 01071B Lb paracasei 1 15 d MRS VAN 25 plusmn 05228B Lb paracasei 1 15 d MRS VAN 25 plusmn 04469B Lb paracasei 1 15 d MRS VAN 24 plusmn 03134C Lb paracasei 1 15 d MRS VAN 23 plusmn 03750D Lb paracasei 10 15 d 2-3ndash6ndash8months M17 30 MRS (30 VAN) 23 plusmn 02576D Lb casei 5 24 h 15 d MRS 30 22 plusmn 07136C Lb paracasei 3 1-2months M17 30 MRS 30 22 plusmn 04158C Lb casei 2 3months MRS VAN 22 plusmn 01475C Lb paracasei 1 1month M17 30 22 plusmn 01451B Lb paracasei 2 15 d MRS 30 21 plusmn 09672D Sc thermophilus 2 2months MRS 30 21 plusmn 01165B Lb paracasei 1 15 d MRS 30 19 plusmn 06938C Lb paracasei 1 2months MRS 30 18 plusmn 04079B Lb paracasei 2 1ndash3months MRS 30 16 plusmn 03952B-C Lb paracasei 5 15 d 2ndash6months MRS (30 VAN) 16 plusmn 03237D Lb paracasei 1 1month MRS VAN 16 plusmn 0154B Lb paracasei 2 2ndash6months M17 30 MRS 30 15 plusmn 01378B Pc pentosaceus 2 1-2months MRS 30 146 plusmn 00853D Lb curvatus 5 1month MRS (30 VAN) 14 plusmn 09814C Pc pentosaceus 2 3months MRS VAN 14 plusmn 08929D Lb coryniformis ssp torquens 2 8months MRS 30 14 plusmn 01135C Lb paracasei 4 1month MRS (30 VAN) 133 plusmn 006632C Lc lactis lactis 4 24 h MRS 30 M17 45 13 plusmn 03731C Lb paracasei 1 2months MRS VAN 13 plusmn 01317C Lb paracasei 4 2ndash8months MRS (30 VAN) 12 plusmn 09474D-C Lb paracasei 1 2months MRS 30 11 plusmn 02255C Pc pentosaceus 2 3months MRS 30 106 plusmn 004443B Pc pentosaceus 2 6months MRS 30 100 plusmn 009923B Lb paracasei 3 15 d 3months MRS VAN 10 plusmn 05467B Lb paracasei 1 1month MRS 30 10 plusmn 0155B Lb paracasei 1 3months MRS 30 10 plusmn 01261C Lb paracasei 2 8months MRS 30 088 plusmn 0081


Table 4 Continued

Cluster Species Isolate number Ripening time Isolation medium and temperature GABA production (mgkg)39C Ln mesenteroides 5 3months MRS (30 VAN) 08 plusmn 06418C Lb paracasei 3 3months MRS (30 VAN) 08 plusmn 02060C Pc pentosaceus 3 15 d 2-3months MRS (30 VAN) 072 plusmn 005819D Lb coryniformis ssp torquens 3 2-3months MRS (30 VAN) 06 plusmn 01295B Sc thermophilus 1 15 d M17 45 06 plusmn 01094B Sc thermophilus 2 15 d 2months M17 45 057 plusmn 007140C Ln mesenteroides 6 1-2months MRS (30 VAN) 057 plusmn 00138C Sc thermophilus 1 15 d M17 45 056 plusmn 004545D-C Ln mesenteroides 18 15 d 1-2-3ndash8months M17 30 MRS (30 VAN) 054 plusmn 00603B Sc thermophilus 2 24 h M17 45 052 plusmn 004126D Lb paracasei 1 8months M17 45 050 plusmn 04283C Sc thermophilus 5 15 d 3months M17 45 050 plusmn 004687C Sc thermophilus 2 24 h M17 45 05 plusmn 02444D-C Ln mesenteroides 5 2ndash6months M17 30 MRS (30 VAN) 048 plusmn 001742B-C Ln mesenteroides 2 1month MRS 30 04 plusmn 01364C Lb plantarum 1 15 d MRS 30 039 plusmn 002763C Ln mesenteroides 2 1month M17 30 037 plusmn 003386D Sc thermophilus 1 3months M17 45 035 plusmn 005546D-C Ln mesenteroides 2 3months M17 30 MRS 30 034 plusmn 00721D Sc thermophilus 2 15 d M17 45 lt0252D Sc thermophilus 1 15 d M17 45 lt0256C Ec faecalis 10 15 d 1-2-3months M17 30-45 lt0257C Sc thermophilus 2 24 h M17 45 lt0259D Lb coryniformis ssp torquens 2 2months M17 30 lt02510C Sc macedonicus 3 3months M17 45 lt02511C Sc macedonicus 4 2months M17 45 lt02512D Sc macedonicus 2 24 h M17 30-45 lt02513D Sc macedonicus 3 8months M17 45 lt02516D Lb acidipiscis 11 6ndash8months MRS (30 VAN) lt02527B Sc thermophilus 1 2months M17 45 lt02530D Ec faecalis 1 2months M17 30 lt02533C Lc lactis cremoris 1 24 h M17 30 lt02541D Ec faecalis 2 2 months M17 30 lt02547C Ln mesenteroides 3 15 d 2months M17 30 lt02548B Lc lactis cremoris 2 1month M17 30 lt02556D Sc macedonicus 2 3months M17 45 lt02557D Ln mesenteroides 2 1month M17 30 lt02581B Lb delbrueckii 1 2months MRS 30 lt02582B Sc thermophilus 5 24 h 1month M17 45 lt02585D Sc thermophilus 3 1month M17 45 lt02588C Sc thermophilus 3 2months M17 45 lt02589C Sc thermophilus 3 24 h M17 30 lt02590B Sc thermophilus 1 24 h M17 45 lt02591B Sc thermophilus 5 1-2months M17 45 lt02592B Sc thermophilus 1 1month M17 45 lt02593B Sc thermophilus 1 1month M17 45 lt02596B Sc thermophilus 3 24 h M17 45 lt02597C Sc thermophilus 1 24 h M17 45 lt025


were the best GABA-producers during the fermentation ofreconstituted skimmedmilk A Lb casei and a Lc lactis subsplactis were used for the manufacture of a GABA-enrichedfermented milk the first strain hydrolyzed milk protein intoglutamic acid and the second converted glutamic acid intoGABA respectively [39] This current study suggests a realpotential of the Sc thermophilus isolate from cluster 84C toproduce GABA in fermented dairy products A daily intakeof fermented milk with an amount of 10mg of GABA for12 weeks has been shown to decrease blood pressure by 174Hg in hypertensive patients [39] Sc thermophilus belongingto the cluster 84C in this current study produces 80mgkgof GABA 125mg of milk fermented with this strain couldtherefore be enough to obtain the daily intake necessary for apotential antihypertensive effect observed by Inoue et al [39]

We are now examining the ability of this Sc thermophilusstrain to produce GABA in fermented milk either alone orin association with other milk protein hydrolyzing LAB andunder simulated gastrointestinal conditions

5 Conclusions

This study describes the diverse lactic microbiota of tra-ditional semihard ldquoNostrano-cheesesrdquo from the Trento alpsin Italy and how this microbiota changes during ripeningWe have also characterised the potential of selected LABisolates to produce GABA under controlled conditions amolecule newly recognised as a putative food bioactiveWe identified one Sc thermophilus strain as a ldquohigh GABAproducerrdquo with considerable biotechnological potential forthe development of new and attractive dairy products animportant commercial objective for increasing the potentialof cheese asmultifunctional dairy product

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This study was financially supported by the project MIROPldquoMetodi innovativi di rintracciabilita di origine e di processoa tutela di produzioni lattiero-casearie tipiche localirdquo withinthe FONDO UNICO DELLA RICERCA of the autonomousprovince of Trento The authors wish to thank the dairyfactories for cheese samples supply

References

[1] M Ortigosa P Barcenas C Arizcun F Perez-Elortondo MAlbisu and P Torre ldquoInfluence of the starter culture on themicrobiological and sensory characteristics of ewersquos cheeserdquoFood Microbiology vol 16 no 3 pp 237ndash247 1999

[2] J T M Wouters E H E Ayad J Hugenholtz and GSmit ldquoMicrobes from raw milk for fermented dairy productsrdquoInternational Dairy Journal vol 12 no 2-3 pp 91ndash109 2002

[3] C Andrighetto F Borney A Barmaz B Stefanon and ALombardi ldquoGenetic diversity of Streptococcus thermophilus

strains isolated from Italian traditional cheesesrdquo InternationalDairy Journal vol 12 no 2-3 pp 141ndash144 2002

[4] L Aquilanti L DellrsquoAquila E Zannini A Zocchetti and FClementi ldquoResident lactic acid bacteria in raw milk CanestratoPugliese cheeserdquo Letters in Applied Microbiology vol 43 no 2pp 161ndash167 2006

[5] G Giraffa C Andrighetto C Antonello et al ldquoGenotypicand phenotypic diversity of Lactobacillus delbrueckii subsplactis strains of dairy originrdquo International Journal of FoodMicrobiology vol 91 no 2 pp 129ndash139 2004

[6] C L Randazzo S Torriani A D L Akkermans W M devos and E E Vaughan ldquoDiversity dynamics and activityof bacterial communities during production of an artisanalsicilian cheese as evaluated by 16S rRNA analysisrdquo Applied andEnvironmental Microbiology vol 68 no 4 pp 1882ndash1892 2002

[7] L Settanni and G Moschetti ldquoNon-starter lactic acid bacteriaused to improve cheese quality and provide health benefitsrdquoFood Microbiology vol 27 no 6 pp 691ndash697 2010

[8] M-C Montel S Buchin A Mallet et al ldquoTraditional cheesesrich and diverse microbiota with associated benefitsrdquo Interna-tional Journal of Food Microbiology vol 177 pp 136ndash154 2014

[9] P Zoon and D Allersma ldquoEye and crack formation in cheeseby carbon dioxide from decarboxylation of glutamic acidrdquoNetherlands Milk and Dairy Journal vol 50 no 2 pp 309ndash3181996

[10] C Jakobs J Jaeken and K M Gibson ldquoInherited disorders ofGABAmetabolismrdquo Journal of Inherited Metabolic Disease vol16 no 4 pp 704ndash715 1993

[11] C G T Wong T Bottiglieri and O C Snead III ldquoGABA120574-hydroxybutyric acid and neurological diseaserdquo Annals ofNeurology vol 54 no 6 pp 3ndash12 2003

[12] S H Oh and C H Oh ldquoBrown rice extracts with enhancedlevels of GABA stimulate immune cellsrdquo Food Science andBiotechnology vol 12 no 3 pp 248ndash252 2003

[13] P Forsythe N Sudo T Dinan V H Taylor and J BienenstockldquoMood and gut feelingsrdquo Brain Behavior and Immunity vol 24no 1 pp 9ndash16 2010

[14] K B Park and S H Oh ldquoIsolation and characterization ofLactobacillus buchneri strains with high gamma-aminobutyricacid producing capacity from naturally aged cheeserdquo FoodScience and Biotechnology vol 15 pp 86ndash90 2006

[15] S Siragusa M de Angelis R di Cagno C G Rizzello R Codaand M Gobbetti ldquoSynthesis of 120574-aminobutyric acid by lacticacid bacteria isolated from a variety of Italian cheesesrdquo Appliedand Environmental Microbiology vol 73 no 22 pp 7283ndash72902007

[16] R Larcher G Nicolini D Bertoldi and L Bontempo ldquoAzotati abasso peso molecolare in formaggi tipicirdquo in Caratterizzazionedi formaggi tipici dellrsquoarco alpino il contributo della ricerca FGasperi and G Versini Eds vol 1 pp 131ndash142 Istituto Agrariodi San Michele allrsquoAdige San Michele Allrsquoadige Italy 2005

[17] E Franciosi L Settanni A Cavazza and E Poznanski ldquoBiodi-versity and technological potential of wild lactic acid bacteriafrom raw cowsrsquo milkrdquo International Dairy Journal vol 19 no 1pp 3ndash11 2009

[18] M S Ammor A Belen Florez and B Mayo ldquoAntibioticresistance in non-enterococcal lactic acid bacteria and bifi-dobacteriardquo FoodMicrobiology vol 24 no 6 pp 559ndash570 2007

[19] T Gregersen ldquoRapid method for distinction of Gram-positivebacteriardquoAppliedMicrobiology and Biothechnology vol 5 no 2pp 123ndash127 1978


[20] E Poznanski A Cavazza F Cappa and P S CocconcellildquoIndigenous rawmilkmicrobiota influences the bacterial devel-opment in traditional cheese from an alpine natural parkrdquoInternational Journal of FoodMicrobiology vol 92 no 2 pp 141ndash151 2004

[21] M Gatti J D D Lindner A De Lorentiis et al ldquoDynamicsof whole and lysed bacterial cells during Parmigiano-Reggianocheese production and ripeningrdquo Applied and EnvironmentalMicrobiology vol 74 no 19 pp 6161ndash6167 2008

[22] S Lick M Keller W Bockelmann and K J Heller ldquoRapididentification of Streptococcus thermophilus by primer-specificPCR amplification based on its lacZ generdquo Systematic andApplied Microbiology vol 19 no 1 pp 74ndash77 1996

[23] C Delorme J-J Godon S D Ehrlich and P Renault ldquoMosaicstructure of large regions of the Lactococcus lactis subspcremoris chromosomerdquoMicrobiology vol 140 no 11 pp 3053ndash3060 1994

[24] L J H Ward andM J Timmins ldquoDifferentiation of Lactobacil-lus casei Lactobacillus paracasei and Lactobacillus rhamnosusby polymerase chain reactionrdquo Letters in Applied Microbiologyvol 29 no 2 pp 90ndash92 1999

[25] A Grifoni M Bazzicalupo C Di Serio S Fancelli and R FanildquoIdentification of Azospirillum strains by restriction fragmentlength polymorphism of the 16S rDNA and of the histidineoperonrdquo FEMS Microbiology Letters vol 127 no 1-2 pp 85ndash911995

[26] M Nomura H Kimoto Y Someya and I Suzuki ldquoNovelcharacteristic for distinguishing Lactococcus lactis subsp lactisfrom subsp cremorisrdquo International Journal of Systematic Bacte-riology vol 49 no 1 pp 163ndash166 1999

[27] P Lehtonen ldquoDetermination of amines and amino acids inwinemdasha reviewrdquo American Journal of Enology and Viticulturevol 47 no 2 pp 127ndash133 1996

[28] P Dolci V Alessandria G Zeppa K Rantsiou and L CocolinldquoMicrobiological characterization of artisanal Raschera PDOcheese analysis of its indigenous lactic acid bacteriardquo FoodMicrobiology vol 25 no 2 pp 392ndash399 2008

[29] P Dolci V Alessandria K Rantsiou L Rolle G Zeppaand L Cocolin ldquoMicrobial dynamics of Castelmagno PDO atraditional Italian cheese with a focus on lactic acid bacteriaecologyrdquo International Journal of FoodMicrobiology vol 122 no3 pp 302ndash311 2008

[30] H Abriouel A Martın-Platero M Maqueda E Valdivia andM Martınez-Bueno ldquoBiodiversity of the microbial communityin a Spanish farmhouse cheese as revealed by culture-dependentand culture-independent methodsrdquo International Journal ofFood Microbiology vol 127 no 3 pp 200ndash208 2008

[31] M G Wilkinson and K N Kilcawley ldquoMechanisms of incor-poration and release of enzymes into cheese during ripeningrdquoInternational Dairy Journal vol 15 no 6ndash9 pp 817ndash830 2005

[32] V L Crow T Coolbear P K Gopal F G Martley L L McKayand H Riepe ldquoThe role of autolysis of lactic acid bacteria in theripening of cheeserdquo International Dairy Journal vol 5 no 8 pp855ndash875 1995

[33] S Tanasupawat O Shida S Okada and K Komagata ldquoLacto-bacillus acidipiscis sp nov and Weissella thailandensis sp novisolated from fermented fish inThailandrdquo International Journalof Systematic and Evolutionary Microbiology vol 50 no 4 pp1479ndash1485 2000

[34] I A Asteri N Robertson D M Kagkli et al ldquoTechnologicaland flavour potential of cultures isolated from traditional Greek

cheesesmdasha pool of novel species and startersrdquo InternationalDairy Journal vol 19 no 10 pp 595ndash604 2009

[35] F Minervini M T Bilancia S Siragusa M Gobbetti and FCaponio ldquoFermented goatsrsquo milk produced with selectedmulti-ple starters as a potentially functional foodrdquo FoodMicrobiologyvol 26 no 6 pp 559ndash564 2009

[36] M Diana A Tres J Quılez M Llombart and M RafecasldquoSpanish cheese screening and selection of lactic acid bacteriawithhigh gamma-aminobutyric acid productionrdquo Food Scienceand Technology vol 56 no 2 pp 351ndash355 2014

[37] H Li and Y Cao ldquoLactic acid bacterial cell factories for gamma-aminobutyric acidrdquo Amino Acids vol 39 no 5 pp 1107ndash11162010

[38] A Ratanaburee D Kantachote W Charernjiratrakul and ASukhoom ldquoSelection of 120574-aminobutyric acid-producing lacticacid bacteria and their potential as probiotics for use as startercultures in Thai fermented sausages (Nham)rdquo InternationalJournal of Food Science and Technology vol 48 no 7 pp 1371ndash1382 2013

[39] K Inoue T Shirai H Ochiai et al ldquoBlood-pressure-loweringeffect of a novel fermentedmilk containing 120574-aminobutyric acid(GABA) in mild hypertensivesrdquo European Journal of ClinicalNutrition vol 57 no 3 pp 490ndash495 2003

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


It has been reported that the use of commercial starters inrawmilk cheesesmaymodify the characteristics of the cheesemicrobiota in particular lowering the microbial biodiversity[1] and it is also well known that mainly LAB microbiotadeveloping during ripening influences the typical organolep-tic characteristics of the cheese [2] Thus LAB represent afundamental process factor for the final attributes and qualityof artisan dairy products such as alpine cheeses Severalstudies have focused on the genotypic and technologicalcharacterization of LAB isolated from different traditionallyfermented cheeses [3ndash6] but little work has so far been doneon ldquoNostrano-cheesesrdquo

In addition to the technological relevance of LAB incheese there is currently much research and industry interestin the potential biological activity of dairy LAB either foruse as probiotics in their own wright or as bioactive agentscapable of modulating the health functionality of cheese andother dairy products [7] Rawmilk cheeses have already beenidentified as a useful source ofmicrobial biodiversity and newLAB strains with health promoting properties [8]

Since caseins are rich in glutamate which is released byproteolytic action the decarboxylation of this amino acidinto 120574-aminobutyric acid (GABA) can have an importanteffect on the formation of eyes in cheese [9] Besidesits technological effect in cheese GABA has several well-characterized physiological functions in mammals includingneurotransmission induction of hypotension diuretic andtranquilizer effects and stimulation of immune cells [10ndash12] Some studies have reported also that GABA derivedfrom the gut may be a neuroactive molecule within thegut-brain axis [13] which is a complex communicationhighway linking the gut environment with both the centraland peripheral nervous systems Strains of Lb buchneri [14]Lb brevis Lb paracasei and Lb plantarum [15] isolatedfrom traditional cheeses have been shown to produce GABAGABA-producing LABhave not been isolated and extensivelycharacterised from traditional alpine cheeses produced inTrento though the presence of GABA in these cheeses hasbeen confirmed and its concentration at the end of ripeningreported at between 120 and 1739mgkg [16] which is highcompared to other Italian cheese varieties (typically 0260to 391mgkg) [15] Therefore the objective of this study wasto analyze the diversity and the successional development ofLAB in traditional ldquoNostrano-cheesesrdquo from the Trento alpsduring cold and summer seasons and to extensively screenand identify GABA-producing LAB isolates

2 Materials and Methods

21 Cheese Factories and Milk Sampling Cheeses were sam-pled in three dairy factories (called B C andD according to aprevious paper [17]) located throughout the Trentino regionand producing traditional alpine cheeses called ldquoNostrano-cheesesrdquo Each factory collected milk from farms within a15 km radius

Two cheese batches from each dairy factory one in Febru-ary and the other in July were sampled making a total of sixbatches subjected to microbiological analyses All factories

processed milk obtained from stabled cows fed with hay dur-ing the ldquocold seasonrdquo from October to May and high moun-tain pasture fed cattle in the summer season from June toSeptember For each of the six batches at least five cheesesamples at different stages of ripening were collected (24hours 15 days 1 month 2 months and 3 months and for fivebatches also 6 and 8 months) making a total of 40 cheesesamples per factory

22 Enumeration and Isolation of Microorganisms Cheesesamples (25 g) were homogenized (2min at 260 rpm) usinga stomacher (laboratory blender stomacher 400 SewardLondon UK) in 225 g peptone water (01 mycologicalpeptone (Oxoid Basingstoke UK)) and serially dilutedDilutions were plated and incubated as follows onto MRSagar acidified to pH55with 5molL lactic acid anaerobicallyfor 2 days at 30∘C and 45∘C for mesophilic and thermophilicrod-shaped LAB respectively onto MRS agar added withvancomycin (8120583gmL Sigma-Aldrich Saint Louis MIUS)[18] and acidified to pH 55 with 5molL lactic acid anaerobi-cally for 72 h at 30∘C for mesophilic heterofermentative rod-shaped LAB onto M17 agar for 2 days aerobically at 30∘Cand anaerobically at 45∘C for mesophilic and thermophiliccoccoid LAB respectively onto KAA aerobically for twodays at 37∘C for enterococci onto PCA added with 10 gLskimmed milk aerobically for 24 h incubated at 30∘C fortotal bacterial count (TBC)All culturemediawere purchasedfrom Oxoid

At least three colonies were picked from each count-able plate Gram-positive colonies (as determined by KOHmethod [19]) and negative to the catalase test (as determinedby transferring fresh colonies from agar medium to a glassslide and adding 5 H

2O2) were isolated Cell morphology

was determined bymicroscopic observation Each isolate waspurified by subsequent culturing onto M17 or MRS and purecultures were stored at minus80∘C in glycerol (20 vv) stocks

23 DNA Extraction and RAPD-PCR DNA was extractedfrom overnight broth cultures of isolated strains Cells werecentrifuged at 10000timesg for 5min and the pellets werewashed twice in sterile distilled water and suspended in1mL of distilled water Cell lysis was achieved using theInstageneMatrix (Bio-Rad Hercules CAUSA) following themanufacturerrsquos instruction

RAPD-PCR was carried out in a total volume of 25120583Lusing primer PC1 [20] Cluster analysis of DNA patternswas carried out using GelCompar II-BioNumerics software(package version 60 Applied Maths Belgium) exploitingthe unweighted pair group method arithmetic averages(UPGMA) Similarity of PCR fingerprinting profiles wascalculated based on Pearson product-moment correlationcoefficient The threshold breakpoint value was fixed to 80isolates with similarity coefficient higher than 80 wereclassified into the same cluster according to Gatti et al [21]

24 Genotypic Identification of LAB One isolate representa-tive of each LAB cluster was genotypically identified by 16SrRNAgene analysis All isolates fromM17 45∘Cwere tested by












3 Results










4 Discussion





34C

64C

9D

86C13D

39C57D

73C-D80B81B

91B

68B

40C

95B

72D

59B58C

26D

3B

31C

56D55C

67B

71B

63C

12D

27B28B

65B66B

94B

43B

49B

8C

32C33C

48B

15C14C

18C

62B

53D

54C

11C10C

87C

88C

47C

38C37C

77B

82C83C

21B-D30D

29D

52B-C

24D25D

78C

79B

76D

61C60C-D

19D20B-C

46C-D

17C

16D

69B70B

97C

96C

50D51B

4B

5B

89B

90B

84D85D

6C7C

36D 35C41D42B-C

74C-D75C

2D1D

92B93B

22B23B

45C-D

44C-D










24 h 12(75)

19(154)

8(80)

18(180)

20(182) 22 (211) 99

(8811)

15 d 8(80) 38 (1028) 15

(114)18

(171)31

(247)27

(225)137

(8255)

1 month 19(109)

43(1528)

42(1923)

31(283)

34(286) 21 (201) 190

(12070)

2 months 28(1612)

54(1719)

49(2039)

36(351)

46(3313)

22(193)

235(14095)

3 months 18(135)

48(2127)

46(2422)

25(241)

23(221)

24(177)

184(12163)

6 monthsa 8(44) 30 (1812) 25

(817)16

(133)22

(220)12

(93)113

(7439)

8 monthsa 15(510)

20(515)

16(412)

25(196)

16(151)

9(54)

101(4848)

TOT 108(6345)

252(101151)

201(84117)

169(15415)

192(16230)

137(11324)

1059(677382)

a119899 = 5









Table3Num

bero

fisolates

andspeciesfou

ndateach

ofthes

amplingtim

eintradition

alalpine

cheeses(119899=6)

Ripening

time

Lb

paracasei

Sc

thermophilus

Ln

mesenteroides

Ec

faecalis

Lc

lactis

Pc

pentosaceus

Lb

acidipiscis

Sc

macedonicu

sLb

casei

Lb

coryniform

isLb

rham

nosus

Lb

curvatus

Lb

plantarum

Lb

delbrueckii

Total

dominant

isolates

24h

mdash19

mdashmdash

6mdash

mdash2

1mdash

mdashmdash

mdash28

15d

208

31

11

mdashmdash

4mdash

2mdash

3mdash

431m

onth

2010

123

22

mdashmdash

mdashmdash

25

mdashmdash

562mon

ths

1410

138

mdash2

mdash4

32

21

mdash1

603mon

ths

153

131

mdash5

mdash5

23

mdashmdash

mdashmdash

476 mon

thsa

12mdash

1mdash

12

6mdash

mdashmdash

mdashmdash

mdashmdash

22

8 mon

thsa

7mdash

3mdash

3mdash

5mdash

mdash2

mdashmdash

mdashmdash

20

TOT

8850

4513

1312

1111

107

66

31

276

a 119899=5





Table 4 Continued





5 Conclusions




Acknowledgments


References


















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology












3 Results










4 Discussion





34C

64C

9D

86C13D

39C57D

73C-D80B81B

91B

68B

40C

95B

72D

59B58C

26D

3B

31C

56D55C

67B

71B

63C

12D

27B28B

65B66B

94B

43B

49B

8C

32C33C

48B

15C14C

18C

62B

53D

54C

11C10C

87C

88C

47C

38C37C

77B

82C83C

21B-D30D

29D

52B-C

24D25D

78C

79B

76D

61C60C-D

19D20B-C

46C-D

17C

16D

69B70B

97C

96C

50D51B

4B

5B

89B

90B

84D85D

6C7C

36D 35C41D42B-C

74C-D75C

2D1D

92B93B

22B23B

45C-D

44C-D










24 h 12(75)

19(154)

8(80)

18(180)

20(182) 22 (211) 99

(8811)

15 d 8(80) 38 (1028) 15

(114)18

(171)31

(247)27

(225)137

(8255)

1 month 19(109)

43(1528)

42(1923)

31(283)

34(286) 21 (201) 190

(12070)

2 months 28(1612)

54(1719)

49(2039)

36(351)

46(3313)

22(193)

235(14095)

3 months 18(135)

48(2127)

46(2422)

25(241)

23(221)

24(177)

184(12163)

6 monthsa 8(44) 30 (1812) 25

(817)16

(133)22

(220)12

(93)113

(7439)

8 monthsa 15(510)

20(515)

16(412)

25(196)

16(151)

9(54)

101(4848)

TOT 108(6345)

252(101151)

201(84117)

169(15415)

192(16230)

137(11324)

1059(677382)

a119899 = 5









Table3Num

bero

fisolates

andspeciesfou

ndateach

ofthes

amplingtim

eintradition

alalpine

cheeses(119899=6)

Ripening

time

Lb

paracasei

Sc

thermophilus

Ln

mesenteroides

Ec

faecalis

Lc

lactis

Pc

pentosaceus

Lb

acidipiscis

Sc

macedonicu

sLb

casei

Lb

coryniform

isLb

rham

nosus

Lb

curvatus

Lb

plantarum

Lb

delbrueckii

Total

dominant

isolates

24h

mdash19

mdashmdash

6mdash

mdash2

1mdash

mdashmdash

mdash28

15d

208

31

11

mdashmdash

4mdash

2mdash

3mdash

431m

onth

2010

123

22

mdashmdash

mdashmdash

25

mdashmdash

562mon

ths

1410

138

mdash2

mdash4

32

21

mdash1

603mon

ths

153

131

mdash5

mdash5

23

mdashmdash

mdashmdash

476 mon

thsa

12mdash

1mdash

12

6mdash

mdashmdash

mdashmdash

mdashmdash

22

8 mon

thsa

7mdash

3mdash

3mdash

5mdash

mdash2

mdashmdash

mdashmdash

20

TOT

8850

4513

1312

1111

107

66

31

276

a 119899=5





Table 4 Continued





5 Conclusions




Acknowledgments


References


















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology









4 Discussion





34C

64C

9D

86C13D

39C57D

73C-D80B81B

91B

68B

40C

95B

72D

59B58C

26D

3B

31C

56D55C

67B

71B

63C

12D

27B28B

65B66B

94B

43B

49B

8C

32C33C

48B

15C14C

18C

62B

53D

54C

11C10C

87C

88C

47C

38C37C

77B

82C83C

21B-D30D

29D

52B-C

24D25D

78C

79B

76D

61C60C-D

19D20B-C

46C-D

17C

16D

69B70B

97C

96C

50D51B

4B

5B

89B

90B

84D85D

6C7C

36D 35C41D42B-C

74C-D75C

2D1D

92B93B

22B23B

45C-D

44C-D










24 h 12(75)

19(154)

8(80)

18(180)

20(182) 22 (211) 99

(8811)

15 d 8(80) 38 (1028) 15

(114)18

(171)31

(247)27

(225)137

(8255)

1 month 19(109)

43(1528)

42(1923)

31(283)

34(286) 21 (201) 190

(12070)

2 months 28(1612)

54(1719)

49(2039)

36(351)

46(3313)

22(193)

235(14095)

3 months 18(135)

48(2127)

46(2422)

25(241)

23(221)

24(177)

184(12163)

6 monthsa 8(44) 30 (1812) 25

(817)16

(133)22

(220)12

(93)113

(7439)

8 monthsa 15(510)

20(515)

16(412)

25(196)

16(151)

9(54)

101(4848)

TOT 108(6345)

252(101151)

201(84117)

169(15415)

192(16230)

137(11324)

1059(677382)

a119899 = 5









Table3Num

bero

fisolates

andspeciesfou

ndateach

ofthes

amplingtim

eintradition

alalpine

cheeses(119899=6)

Ripening

time

Lb

paracasei

Sc

thermophilus

Ln

mesenteroides

Ec

faecalis

Lc

lactis

Pc

pentosaceus

Lb

acidipiscis

Sc

macedonicu

sLb

casei

Lb

coryniform

isLb

rham

nosus

Lb

curvatus

Lb

plantarum

Lb

delbrueckii

Total

dominant

isolates

24h

mdash19

mdashmdash

6mdash

mdash2

1mdash

mdashmdash

mdash28

15d

208

31

11

mdashmdash

4mdash

2mdash

3mdash

431m

onth

2010

123

22

mdashmdash

mdashmdash

25

mdashmdash

562mon

ths

1410

138

mdash2

mdash4

32

21

mdash1

603mon

ths

153

131

mdash5

mdash5

23

mdashmdash

mdashmdash

476 mon

thsa

12mdash

1mdash

12

6mdash

mdashmdash

mdashmdash

mdashmdash

22

8 mon

thsa

7mdash

3mdash

3mdash

5mdash

mdash2

mdashmdash

mdashmdash

20

TOT

8850

4513

1312

1111

107

66

31

276

a 119899=5





Table 4 Continued





5 Conclusions




Acknowledgments


References


















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


34C

64C

9D

86C13D

39C57D

73C-D80B81B

91B

68B

40C

95B

72D

59B58C

26D

3B

31C

56D55C

67B

71B

63C

12D

27B28B

65B66B

94B

43B

49B

8C

32C33C

48B

15C14C

18C

62B

53D

54C

11C10C

87C

88C

47C

38C37C

77B

82C83C

21B-D30D

29D

52B-C

24D25D

78C

79B

76D

61C60C-D

19D20B-C

46C-D

17C

16D

69B70B

97C

96C

50D51B

4B

5B

89B

90B

84D85D

6C7C

36D 35C41D42B-C

74C-D75C

2D1D

92B93B

22B23B

45C-D

44C-D










24 h 12(75)

19(154)

8(80)

18(180)

20(182) 22 (211) 99

(8811)

15 d 8(80) 38 (1028) 15

(114)18

(171)31

(247)27

(225)137

(8255)

1 month 19(109)

43(1528)

42(1923)

31(283)

34(286) 21 (201) 190

(12070)

2 months 28(1612)

54(1719)

49(2039)

36(351)

46(3313)

22(193)

235(14095)

3 months 18(135)

48(2127)

46(2422)

25(241)

23(221)

24(177)

184(12163)

6 monthsa 8(44) 30 (1812) 25

(817)16

(133)22

(220)12

(93)113

(7439)

8 monthsa 15(510)

20(515)

16(412)

25(196)

16(151)

9(54)

101(4848)

TOT 108(6345)

252(101151)

201(84117)

169(15415)

192(16230)

137(11324)

1059(677382)

a119899 = 5









Table3Num

bero

fisolates

andspeciesfou

ndateach

ofthes

amplingtim

eintradition

alalpine

cheeses(119899=6)

Ripening

time

Lb

paracasei

Sc

thermophilus

Ln

mesenteroides

Ec

faecalis

Lc

lactis

Pc

pentosaceus

Lb

acidipiscis

Sc

macedonicu

sLb

casei

Lb

coryniform

isLb

rham

nosus

Lb

curvatus

Lb

plantarum

Lb

delbrueckii

Total

dominant

isolates

24h

mdash19

mdashmdash

6mdash

mdash2

1mdash

mdashmdash

mdash28

15d

208

31

11

mdashmdash

4mdash

2mdash

3mdash

431m

onth

2010

123

22

mdashmdash

mdashmdash

25

mdashmdash

562mon

ths

1410

138

mdash2

mdash4

32

21

mdash1

603mon

ths

153

131

mdash5

mdash5

23

mdashmdash

mdashmdash

476 mon

thsa

12mdash

1mdash

12

6mdash

mdashmdash

mdashmdash

mdashmdash

22

8 mon

thsa

7mdash

3mdash

3mdash

5mdash

mdash2

mdashmdash

mdashmdash

20

TOT

8850

4513

1312

1111

107

66

31

276

a 119899=5





Table 4 Continued





5 Conclusions




Acknowledgments


References


















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




24 h 12(75)

19(154)

8(80)

18(180)

20(182) 22 (211) 99

(8811)

15 d 8(80) 38 (1028) 15

(114)18

(171)31

(247)27

(225)137

(8255)

1 month 19(109)

43(1528)

42(1923)

31(283)

34(286) 21 (201) 190

(12070)

2 months 28(1612)

54(1719)

49(2039)

36(351)

46(3313)

22(193)

235(14095)

3 months 18(135)

48(2127)

46(2422)

25(241)

23(221)

24(177)

184(12163)

6 monthsa 8(44) 30 (1812) 25

(817)16

(133)22

(220)12

(93)113

(7439)

8 monthsa 15(510)

20(515)

16(412)

25(196)

16(151)

9(54)

101(4848)

TOT 108(6345)

252(101151)

201(84117)

169(15415)

192(16230)

137(11324)

1059(677382)

a119899 = 5









Table3Num

bero

fisolates

andspeciesfou

ndateach

ofthes

amplingtim

eintradition

alalpine

cheeses(119899=6)

Ripening

time

Lb

paracasei

Sc

thermophilus

Ln

mesenteroides

Ec

faecalis

Lc

lactis

Pc

pentosaceus

Lb

acidipiscis

Sc

macedonicu

sLb

casei

Lb

coryniform

isLb

rham

nosus

Lb

curvatus

Lb

plantarum

Lb

delbrueckii

Total

dominant

isolates

24h

mdash19

mdashmdash

6mdash

mdash2

1mdash

mdashmdash

mdash28

15d

208

31

11

mdashmdash

4mdash

2mdash

3mdash

431m

onth

2010

123

22

mdashmdash

mdashmdash

25

mdashmdash

562mon

ths

1410

138

mdash2

mdash4

32

21

mdash1

603mon

ths

153

131

mdash5

mdash5

23

mdashmdash

mdashmdash

476 mon

thsa

12mdash

1mdash

12

6mdash

mdashmdash

mdashmdash

mdashmdash

22

8 mon

thsa

7mdash

3mdash

3mdash

5mdash

mdash2

mdashmdash

mdashmdash

20

TOT

8850

4513

1312

1111

107

66

31

276

a 119899=5





Table 4 Continued





5 Conclusions




Acknowledgments


References


















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Table3Num

bero

fisolates

andspeciesfou

ndateach

ofthes

amplingtim

eintradition

alalpine

cheeses(119899=6)

Ripening

time

Lb

paracasei

Sc

thermophilus

Ln

mesenteroides

Ec

faecalis

Lc

lactis

Pc

pentosaceus

Lb

acidipiscis

Sc

macedonicu

sLb

casei

Lb

coryniform

isLb

rham

nosus

Lb

curvatus

Lb

plantarum

Lb

delbrueckii

Total

dominant

isolates

24h

mdash19

mdashmdash

6mdash

mdash2

1mdash

mdashmdash

mdash28

15d

208

31

11

mdashmdash

4mdash

2mdash

3mdash

431m

onth

2010

123

22

mdashmdash

mdashmdash

25

mdashmdash

562mon

ths

1410

138

mdash2

mdash4

32

21

mdash1

603mon

ths

153

131

mdash5

mdash5

23

mdashmdash

mdashmdash

476 mon

thsa

12mdash

1mdash

12

6mdash

mdashmdash

mdashmdash

mdashmdash

22

8 mon

thsa

7mdash

3mdash

3mdash

5mdash

mdash2

mdashmdash

mdashmdash

20

TOT

8850

4513

1312

1111

107

66

31

276

a 119899=5





Table 4 Continued





5 Conclusions




Acknowledgments


References


















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





Table 4 Continued





5 Conclusions




Acknowledgments


References


















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Table 4 Continued





5 Conclusions




Acknowledgments


References


















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




5 Conclusions




Acknowledgments


References


















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Research Article Biodiversity and -Aminobutyric Acid ...downloads.hindawi.com › journals › bmri › 2015 › 625740.pdfResearch Article Biodiversity and -Aminobutyric Acid Production