Characterization of the Microbial Diversity and ChemicalComposition of Gouda Cheese Made by Potential Probiotic Strainsas an Adjunct Starter CultureNam Su Oh,† Jae Yeon Joung,†,§ Ji Young Lee,†,§ Sae Hun Kim,*,§ and Younghoon Kim*,#

†R&D Center, Seoul Dairy Cooperative, Ansan, Kyunggi 15407, South Korea§Department of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul 02841, South Korea#Department of Animal Science and Institute of Milk Genomics, Chonbuk National University, Jeonju 54896, South Korea

ABSTRACT: This study characterized the microbial diversity and chemical properties of Gouda cheese made by probioticsduring ripening periods. Lactobacillus plantarum H4 (H4) and Lactobacillus fermentum H9 (H9), which demonstrate probioticproperties and bioactivity, were used as adjunct starter cultures. Gouda cheese made with H4 (GCP1) and H9 (GCP2)demonstrated the highest production of formic acid and propionic acid, respectively. Moreover, the bacterial diversity, includingrichness and evenness of nonstarter lactic acid bacteria (NSLAB), increased in probiotic cheeses. Specifically, Lactobacillus,Leuconostoc, and Streptococcaceae were present at higher concentrations in probiotic cheeses than in control Gouda cheese(GCC). The proportion of H4 in GCP1 increased and culminated at 1.76%, whereas H9 in GCP2 decreased during ripening.Peptide profiles were altered by the addition of probiotics and included various bioactive peptides. In particular, three peptidefragments are newly detected. Therefore, Gouda cheese could be used as an effective probiotic carrier for H4 and H9.

KEYWORDS: Gouda cheese, adjunct cultures, probiotics, microbial community, organic acid, peptide profiling

■ INTRODUCTION

Gouda cheese is produced from pasteurized milk acidified bymesophilic lactic acid bacteria (LAB) such as Lactococcus lactissubsp. lactis, Lc. lactis subsp. cremoris, Lc. lactis subsp. lactis biovardiacetylactis, and Leuconostoc mesenteroides subsp. cremoris.1

Gouda cheese is generally matured for 2−3 months atapproximately 13−15 °C.2 Adjunct cultures of Lactobacillushave been studied to control the ripening process and the growthof the microbial composition of nonstarter LAB (NSLAB)in Cheddar cheese,3 Manchego cheese,4 and Gouda cheese5

manufacturing, as well as to improve organoleptic properties.Adjunct cultures can change the microbial community ofNSLAB, and this coincides with cheese proteolysis and lipolysisduring ripening.6 Moreover, cheese provides a valuablealternative to fermented milks and yogurts as a probiotic foodcarrier by incorporating probiotics in cheese as adjunct cultures.Probiotic food products including probiotic cheese mustdemonstrate their efficacy and maintain the probiotic viabilityin the final products.7 The incorporation of probiotics should notimply a loss of quality of products.7 Beneficial effects on healthrelated to probiotic cheese have been reported; probiotic freshcheese containing Lb. acidophilus, B. bif idum, and Lb. paracaseidemonstrated immune-modulating capacity in mice,8 andprobiotic Edam cheese containing Lb. rhamnosus was studiedwith regard to the risk of dental caries.9 However, there are fewstudies demonstrating healthy functional Gouda cheese withprobiotic adjunct cultures. The use of bacterial strains with healthbenefits could be a possible strategy for the cheese industry toaddress the increased demand for new or special cheeses. Next-generation sequencing (NGS) methodologies provide a powerfultool to analyze complex microbial communities of fermentedfood materials, because multiplex barcoded pyrosequencing allows

the analysis of multiple samples in a single run inexpensively.10,11

A previous study reported that Cheddar cheese made withprobiotics strains of Lactobacillus isolated from the human smallintestine has been characterized for its probiotic potential. Theprobiotic strains were sustained at a high viability in cheeseduring up to 120 days of ripening.12 In this study, Lactobacillusplantarum H4 (H4) and Lactobacillus fermentum H9 (H9),isolated from infant feces, were selected as probiotic adjunctcultures. The strains exhibited probiotic potential such as acidand bile tolerance, proteolytic activity, adhesion to intestine,and additional bioactivities such as antioxidant and cholesterol-lowering activities.13,14 The main objective of this study wasto investigate the effects of probiotics as adjunct cultures on theripening of Gouda cheese. Accordingly, the microbial communi-ties and chemical properties were analyzed.

■ MATERIALS AND METHODSBacterial Culture. Freeze-dried direct vat starter culture (DVS)

Lc. lactis subsp. cremoris, Leuconostoc, Lc. lactis subsp. lactis, andLc. lactis subsp. lactis biovar diacetylactis with code CHN-11 wereobtained from Chr. Hansen (Horsholm, Denmark). H4 and H9probiotic cultures were selected for this study on the basis of theirprobiotic potential and bioactivities. For initial strain selection,Lactobacillus strains were isolated from plants and human feces.Briefly, the plant and feces samples were weighed and homogenizedfor 30 s in saline and diluted. Aliquots of serial dilutions were plated onde Man, Rogosa, and Sharpe (MRS) agar (Difco Laboratories, Detroit,MI, USA) and incubated at 37 °C for 48−72 h. In total, 450 strains

Received: June 15, 2016Revised: August 31, 2016Accepted: September 8, 2016Published: September 8, 2016

Article

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© 2016 American Chemical Society 7357 DOI: 10.1021/acs.jafc.6b02689J. Agric. Food Chem. 2016, 64, 7357−7366

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were isolated and their probiotic potential was evaluated, using varioustests such as acid and bile tolerance, adhesion to intestine, proteinhydrolysis activity, antioxidant activity, and cholesterol-reducingability (data not shown).13−15 Then, we performed complete genomesequencing of two selected probiotic strains; the bioproject numbersfor Lb. plantarum H4 and Lb. fermentum H9 have been depositedin the NCBI GenBank (http://www.ncbi.nlm.nih.gov/genbank/) asaccession numbers PRJNA325681 and PRJNA325680, respectively.Manufacture of Probiotic Gouda Cheese. Three separate trials

of Gouda cheese manufacturing were performed with or withoutprobiotics. Raw milk was obtained from Seoul Dairy Cooperative(Ansan, Kyunggi, Korea) to make Gouda cheese. Probiotic Goudacheese was manufactured in a pilot scale cheese vat. Specifically, 50 kgof raw milk was pasteurized at 72 °C for 15 s and cooled to 34 °C.The pasteurized milk was inoculated with 0.016% of starter cultureCHN-11 (Chr. Hansen, Horsholm, Denmark) and 0.01% of probiotics(H4 and H9, 6.35 ± 0.02 and 7.22 ± 0.02 log CFU/mL, respectively),and 0.01% of CaCl2 was added. The species that compose starterculture CHN-11 were Lactococcus lactis subsp. cremoris, Leuconostoc,Lactococcus lactis subsp. lactis, and Lactococcus lactis subsp. lactisbiovar diacetylactis. When the acidity increased by 0.01% (after 40 min,0.1422 → 0.1520%), 0.02% rennet (Chr. Hansen, New Zealand) wasadded to induce the formation of curds. The milk was allowed tocoagulate for 60 min, and the coagulum was then cut into 9 mm cube-shaped particles. After the curds were simultaneously stirred for30 min, the whey was removed (30% of the volume of the milk) andwater (20% of the volume of the milk) at 38 °C was added. The curdswere stirred for 20 min. Following stirring, 30% (the volume of themilk) of whey was removed and 20% (the volume of the milk) of73 °C water was added (2.5 kg/5 min) to the stirring curds (for20 min), as the temperature of the curd−whey mixture graduallyincreased from 34 to 37 °C. The whey was drained, and the curd wasput into a mold and prepressed for 60 min. The curd was pressed(for 12 h) until the pH reached 5.05−5.10. The probiotic Goudacheese was removed from the mold and soaked in a 20% saturatedbrine solution at 5 °C for 24 h. The cheese was allowed to dry for2 days. Control Gouda cheese was manufactured by the samemanufacturing process as probiotic cheese but without supplementa-tion of probiotics. The Gouda cheese was packed in cheese wax andripened for 8 weeks at 15 °C. Samples of Gouda cheese were collectedfor analysis every 2 weeks post manufacture.Chemical Compositions of Gouda Cheese. Gouda cheeses

were grated and analyzed in triplicate for protein, fat, lactose, moisture,pH, salt in moisture (S/M), fat in dry matter (FDM), and moisturein nonfat substrates (MNFS) as described by the Association ofOfficial Analytical Chemists, chapters 33.2 and 33.7.16 Organic acid wasanalyzed with HPLC-UVD according to the method of Ong et al.17

with a slight modification in which the concentration of sulfuricacid used as mobile phase and extraction buffer was changed from0.009 to 0.005 N.PCR Amplification and Pyrosequencing. PCR amplification

was performed using primers targeting from V1 to V3 regions of the16S rRNA gene with extracted DNA from 15 different cheese samplesper the method of Riquelme.10 For bacterial amplification, barcodedprimers of 27F 5′-CCTATCCCCTGTGTGCCTTGGCAGTCT-CAGACGAGTTTGATCMTGGCTCAG-3′ (underlined sequenceindicates the target region primer) and 518R 5′-CCATCTCATCCC-TGCGTGTCTCCGACTCAG-X-AC-WTTACCGCGGCTGCTGG-3′ (“X” indicates the unique barcode for each subject) (http://oklbb.ezbiocloud.net/content/1001). The amplifications were carried outunder the following conditions: initial denaturation at 95 °C for5 min, followed by 30 cycles of denaturation at 95 °C for 30 s, primerannealing at 55 °C for 30 s, and extension at 72 °C for 30 s, with afinal elongation at 72 °C for 5 min. The PCR product was confirmedby using 2% agarose gel electrophoresis and visualized under a GelDoc system (BioRad, Hercules, CA, USA). The amplified productswere purified with the QIAquick PCR purification kit (Qiagen,Valencia, CA, USA). Equal concentrations of purified products werepooled together and short fragments (nontarget products) removedwith Ampure beads kit (Agencourt Bioscience, Beverly, MA, USA).

The quality and product size were assessed on a Bioanalyzer 2100(Agilent, Palo Alto, CA, USA) using a DNA 7500 chip. Mixedamplicons were conducted by emulsion PCR and then depositedon Picotiter plates. The sequencing was carried out at Chunlab, Inc.(Seoul, Korea), with GS Junior Sequencing system (Roche, Branford,CT, USA) according to the manufacturer’s instructions.

Pyrosequencing Data Analysis. The basic analysis wasconducted according to the previous descriptions in other studies.18−20

Obtained reads from the different samples were sorted by uniquebarcodes of each PCR product. The sequences of the barcode, linker,and primers were removed from the original sequencing reads. Anyreads containing two or more ambiguous nucleotides, low quality score(average score < 25), or reads shorter than 300 bp were discarded.Potential chimera sequences were detected by the bellerophone method,which compares the BLASTN search results between forward-halfand reverse-half sequences.21 After removal of chimera sequences, thetaxonomic classification of each read was assigned against the EzTaxon-edatabase (http://eztaxon-e.ezbiocloud.net),22 which contains 16S rRNAgene sequence of type strains that have valid published names andrepresentative species level phylotypes of either cultured or unculturedentries in the GenBank database with complete hierarchical taxonomicclassification from the phylum to the species. The richness and diversityof samples were determined by Chao1 estimation and Shannon diversityindex at the 3% distance. Random subsampling was conducted to equalizeread size of samples for comparing different read sizes among samples.The pyrosequencing data were analyzed using the CLcommunityprogram (Chunlab Inc., Seoul, Korea).

Peptide Profiling by MALDI-TOF/MS/MS. The water-solublenitrogen (WSN) of cheese samples was prepared with the methodof Ardo and Polychroniadou23 with slight modification. WSN wasextracted from cheese four times and freeze-dried. The cheese peptideanalysis was performed according to the method of Oh et al.24 withMALDI-TOF/MS using Bruker Autoflex (Bruker Daltonics, Bremen,Germany).

Statistical Analysis. All data were expressed as means ± SD.Statistical significance for the differences between the groups wasassessed using Duncan’s multiple-range tests. IBM SPSS statisticssoftware version 22 (IBM Corp., Armonk, NY, USA) was used toperform all statistical tests. Values of P < 0.05 were considered toindicate a significant difference.

■ RESULTSChemical Composition of Gouda Cheese. The chemical

composition of Gouda cheese is shown in Table 1. No signifi-cant differences in the chemical compositions such as thecontents of protein, fat, moisture, and S/M of three types ofGouda cheese were observed. However, the amount of lactosewas lowest in Gouda cheese made with H9 (GCP2) at theinitial stages of ripening, but reduced entirely in all cheesesamples. The moisture content was also significantly reducedafter ripening, with a slight increase in the protein, fat, and S/Mcontent of the cheeses. The lactose metabolic activity of themicroorganisms in Gouda cheese was assessed by estimatingthe production of organic acids such as citric acid, pyruvic acid,lactic acid, acetic acid, propionic acid, and formic acid (Table 2).The concentration of organic acids was significantly affected bythe addition of probiotics and ripening time. The main organicacids in Gouda cheese throughout ripening were citric, lactic,and acetic acids. An increase in concentration during ripeningwas observed in pyruvic and propionic acids, whereas the levelsof lactic and acetic acids were irregular, demonstrating a slightdecreasing tendency. The concentration of citric acid increasedin the early stages of ripening and eventually decreased. Inaddition, the disappearance rate of citric, lactic, and acetic acidsin probiotic cheeses was significantly higher than for GCC.Propionic and formic acids were hardly detected in GCC, andthe level of propionic acid exponentially increased in Gouda

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cheese made with H4 (GCP1) and GCP2 at 6 and 4 weeks ofripening, respectively. The level of formic acid in GCP2 washighest at the beginning of ripening (week 0), but decreasedduring the ripening period. However, it gradually increased inGCP1 for 6 weeks of ripening, with the highest levels observedat the end of the ripening period.Comparison of Species and Diversity Estimates of

Bacterial Community in Gouda Cheese. Samples of Goudacheese made with probiotics collected at 0, 2, 4, 6, and 8 weeksof ripening were analyzed for microbial diversity by high-throughput sequencing (Table 3). A total of 110,679 bacterialsequencing reads with an average sequence length of 397.94 bpand an average of 7379 sequencing reads per sample wereobtained from three types of Gouda cheese. Good’s coverageindex, an estimator of sampling completeness, for each data setwas >99% in all samples, indicating that the rarified sequencingdepth was sufficient to evaluate the bacterial diversity of Goudacheese.25 The rarefaction curves (data not shown) were approxi-mately an asymptote, which indicated that the analyzed datasufficiently reflected the bacterial community and the entirebacterial diversity of cheese samples. The alpha diversity analysisrevealed that Chao 1 and Ace estimators, which provide therichness of bacterial species in a sample, decreased duringthe first 2 weeks of ripening, increased until week 6, and then

decreased again until the end of ripening in all cheese samples.In particular, GCP1 and GCP2 made with probiotics showedhigher values of bacterial richness parameters than GCC. More-over, Shannon’s diversity index revealed higher diversity inprobiotic cheeses, which indicated that the evenness of bacterialspecies distribution in GCP1 and GCP2 was higher than in GCC.

Bacterial Communities of Gouda Cheese. The 16S rRNAgene sequencing reads were classified into different taxonomies.The relative abundance of each sample was generated intophylum, order, family, genus, and species levels (Table 4;Figures 1 and 2). At the phylum level, Firmicutes dominated,and Proteobacteria was identified only in GCP2 at week 8 ofripening. At the family level, all cheese samples were dominatedby the family Streptococcaceae (>96.4%); however, the secondmost abundant family varied among samples. As shown inTable 4, Leuconostocaceae was the only family group (exceptStreptococcaceae) present in GCC, with an increase in propor-tion from 0.28 to 0.88% during ripening. GCP1 containedLeuconostocaceae (1.21%) and Lactobacillaceae (1.13%) as thesecond dominant family with similar proportions. The propor-tion of Leuconostocaceae increased until week 4 of ripeningand then decreased, whereas Lactobacillaceae increased throughthe ripening period from 0.35 to 1.81%. The Lactobacillaceaefamily was the second most abundant group, of which the

Table 2. Organic Acid Level of Gouda Cheese Made with a Probiotics Adjunct Culture during the Ripening Perioda

lactic acid(g/kg of cheese)

citric acid(mg/kg of cheese)

acetic acid(mg/kg of cheese)

pyruvic acid(mg/kg of cheese)

propionic acid(mg/kg of cheese)

formic acid(mg/kg of cheese)

GCC1b 0 weeks 26.6 ± 0.3ab 452.5 ± 3.3h 795.7 ± 13.7a 41.8 ± 0.3i ND 7.8 ± 0.1h2 weeks 26.1 ± 0.1c 763.1 ± 26.5d 660.3 ± 1.6g 53.9 ± 0.1i ND ND4 weeks 24.2 ± 0.1e 916.2 ± 32.9b 675.9 ± 6.1f 106.9 ± 0.8g 1.4 ± 0.1e ND6 weeks 25.6 ± 0.1d 1066.6 ± 0.5a 751.2 ± 5.2c 161.0 ± 0.6e 18.4 ± 3.4d ND8 weeks 24.4 ± 0.1e 1044.9 ± 19.3a 712.1 ± 0.1e 179.2 ± 0.5de ND ND

GCP1 0 weeks 26.7 ± 0.4a 594.5 ± 14.6f 720.0 ± 4.8de 78.2 ± 4.0h 15.2 ± 1.0d 11.0 ± 0.1g2 weeks 26.7 ± 0.1a 762.1 ± 26.8d 785.3 ± 1.8a 87.5 ± 2.1h 12.4 ± 1.0e 22.0 ± 0.4d4 wk 26.2 ± 0.1bc 905.3 ± 13.8b 767.4 ± 1.2b 129.7 ± 0.8g 19.1 ± 1.2d 38.3 ± 0.2c6 weeks 21.7 ± 0.2d 749.1 ± 19.5d 646.6 ± 7.3g 220.5 ± 3.3b 118.5 ± 9.1c 51.9 ± 1.7a8 weeks 22.8 ± 0.2e 852.4 ± 18.3c 710.3 ± 0.1e 195.9 ± 1.8c 129.1 ± 8.0b 44.7 ± 1.8b

GCP2 0 weeks 24.5 ± 0.2e 406.4 ± 2.6i 733.4 ± 5.7d 85.7 ± 1.2h ND 21.9 ± 0.3d2 weeks 25.3 ± 0.3c 639.0 ± 0.6e 689.2 ± 2.5f 102.7 ± 4.5g 16.3 ± 2.7d 19.7 ± 2.1e4 weeks 21.0 ± 0.1h 469.3 ± 19.3h 614.7 ± 0.6h 172.3 ± 11.6d 129.8 ± 4.4b 20.6 ± 1.2de6 weeks 19.9 ± 0.3i 477.4 ± 12.1h 485.7 ± 7.0i 196.5 ± 16.6c 134.4 ± 6.1b 13.8 ± 0.1f8 weeks 19.2 ± 0.1j 529.7 ± 26.2g 489.4 ± 16.7i 233.4 ± 8.2a 177.8 ± 2.6a 9.9 ± 1.4g

aValues are presented as the mean ± SD (n = 3). Data followed by a different lower case letter within a column were significantly different(P < 0.05). bGCC, Gouda cheese control; GCP1, Gouda cheese made with a probiotics 1 (Lb. plantarum H4) adjunct culture; GCP2, Gouda cheesemade with a probiotics 2 (Lb. fermentum H9) adjunct culture.

Table 1. Chemical Compositions (Percent) of Gouda Cheesea

protein fat lactose moisture S/Mb FDM MNFS

GCCc 0 weeks 24.94 ± 0.53b 30.12 ± 0.50a 0.026 ± 0.001a 42.23 ± 0.23a 3.04 ± 0.01b 52.15 ± 0.87a 60.44 ± 0.33a8 weeks 25.50 ± 0.12a 30.65 ± 0.69a ND 40.06 ± 0.25b 4.37 ± 0.17a 51.13 ± 1.16a 58.27 ± 0.35b

GCP1 0 weeks 24.87 ± 0.19b 30.22 ± 0.36a 0.024 ± 0.001a 42.17 ± 0.79a 3.01 ± 0.07b 53.33 ± 0.89a 60.43 ± 1.14a8 weeks 25.34 ± 0.16ab 30.54 ± 0.33a ND 40.24 ± 0.39b 4.33 ± 0.11a 51.96 ± 1.76a 57.92 ± 0.56b

GCP2 0 weeks 24.93 ± 0.10b 30.35 ± 0.70a 0.018 ± 0.000b 42.81 ± 0.50a 2.99 ± 0.10b 53.08 ± 1.22a 61.47 ± 0.72a8 weeks 25.52 ± 0.16a 30.57 ± 0.52a ND 40.71 ± 0.08b 4.33 ± 0.00a 51.56 ± 0.88a 58.63 ± 0.12b

aValues are presented as the mean ± SD (n = 3). Data followed by a different lower case letter within a column are significantly different (P < 0.05).bS/M, salt in moisture; FDM, fat in dry matter; MNFS, moisture in nonfat substance. cGCC, Gouda cheese control; GCP1, Gouda cheese madewith a probiotics 1 (Lb. plantarum H4) adjunct culture; GCP2, Gouda cheese made with a probiotics 2 (Lb. fermentum H9) adjunct culture.

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proportion was approximately 2-fold higher than that ofLeuconostocaceae in GCP2. Notably, Xanthomonadaceae,which belongs to the phylum Proteobacteria, was detectedonly in GCP2. Likewise, further classification to genus levelindicated that bacterial communities varied considerably amongthe three types of cheese samples during ripening. Specifically,probiotic cheeses showed a more diverse bacterial communitythan GCC.Pyrosequencing revealed that five genera (relative abundance

> 0.01%) of Lactococcus, Streptococcus, Leuconostoc, Lactobacillus,and Stenotrophomonas were identified in Gouda cheese. Themost abundant genus was Lactococcus, with >96.1% abundancein all cheese samples. The second dominant genus differed bythe type of starter culture used for the manufacture of Goudacheese: Leuconostoc for GCC, Leuconostoc and Lactobacillus forGCP1, and Lactobacillus for GCP2. Lactobacillus, unclassifiedLactobacillaceae, and Stenotrophomonas were detected onlyin probiotic cheeses. A sequence that could not be assignedtaxonomic affiliations at a 97% level of similarity was labeled“unclassified”.The taxonomic classification of cheese bacteria at species

level is presented, divided into SLAB and NSLAB in Figures 1and 2, respectively. Lc. lactis subsp. cremoris and Lc. lactis groupscomprised most (94.3−99.9%) of the microbial community inGouda cheese. In particular, Lc. lactis subsp. cremoris tended todecrease with ripening, in GCP1 and GCP2. Figure 2 showsthe dynamic and diverse NSLAB bacterial community. In GCC,the proportion of Lactococcus decreased, whereas that of Ln.pseudomesenteroides increased, as ripening progressed. GCP1showed a similar trend for Lactococcus and Ln. pseudomesenter-oides proportions compared to GCC, except that Lb. plantarumwas specifically detected, and the proportion increasedthroughout ripening from 0.34 to 1.76%. Lb. fermentum wasidentified only in GCP2, although the proportion dramaticallyincreased initially and then gradually decreased until week 8 ofripening. Unclassified Lactobacillus, Lactobacillales, and Lacto-bacillaceae were present only in probiotic cheeses. Moreover,unclassified Lactococcus and unclassified Streptococcaceae werepresent at higher proportions in GCP1 and GCP2 than in GCC.

The results of the microbial community and diversity analysesbased on taxonomy classification at species level correspondedwith the results of the heat map analysis of microbialcommunities in cheese samples, which were distributed on thebasis of the six main species (Figure 3). Each cheese sample,based on the extent of ripening, contained a sample-specificbacterial community. The unweighted pair group method witharithmetic mean (UPGMA) tree constructed on the basis ofthe dissimilarity of the bacterial community in cheese samplesindicated two clusters as group 1 (GCP1 6 week, GCP1 4 week,GCP2 6 week, GCP2 8 week, GCP2 4 week, GCP1 8 week,GCP2 2 week, and GCP2 0 week) and group 2 (GCC 8 week,GCC 4 week, GCC 6 week, GCP1 2 week, GCC 2 week, GCC0 week, and GCP1 0 week). Cheese samples manufactured fromthe same starter culture clustered together, and samples withsimilar ripening degree clustered closely.

Statistical Comparison of the Microbial CommunityStructure of Gouda Cheese. Canonical correspondenceanalysis (CCA) results for several microbial assemblages inrelation to chemical compositions of Gouda cheese and organicacids are shown in Figure 4. The A and B triplot shows therelationship between the type of starter culture, bacterialcomposition, and chemical composition (protein, fat, lactose,moisture, S/M, FDM, MNFS, and organic acids). The CCAtriplot indicated that several properties, including lactose, citricacid, formic acid, propionic acid, and lactic acid contents, wereimportant for bacterial growth and community formation.These factors also had an effect on the distribution of Goudacheese clusters according to probiotics and cheese ripening.In contrast, the chemical compositions analysis (Table 1),which is the CCA triplot for chemical compositions, showed nodiscernible trend. However, the triplot analysis for organic acidshowed that formic acid and propionic acid were consideredthe key factors for the distribution of cheese samples accordingto ripening period. Lactobacillus, Leuconostoc, and Streptococ-caceae highly correlated with formic, pyruvic, and propionicacid content. The cheese samples were distributed in threegroups by probiotics; particularly probiotic cheeses at initialand late ripening were clustered separately. This indicates that

Table 3. Number of Sequences, Observed Diversity Richness (OTUs), and Diversity Estimates of Bacteria in Gouda Cheeseduring Ripening Period

no. of seq OTUs Chao 1 Ace Shannon Simpson

GCCa 0 weeks 7622 60 62 61.87 2.96 0.102 weeks 8466 48 49 49.49 2.85 0.094 weeks 8542 59 61 60.59 2.94 0.086 weeks 7773 69 80 81.25 2.76 0.128 weeks 7828 56 63 62.01 2.97 0.09

GCP1 0 weeks 7961 80 84 84.65 2.89 0.102 weeks 7236 68 70 71.22 3.08 0.074 weeks 6985 85 88 88.51 3.35 0.056 weeks 6627 88 97 96.63 3.32 0.078 weeks 5854 68 73 76.85 3.17 0.06

GCP2 0 weeks 7547 67 79 72.56 3.07 0.072 weeks 6709 71 82 77.59 3.09 0.074 weeks 7085 86 97 93.47 3.33 0.066 weeks 7262 82 93 92.73 3.13 0.078 weeks 7182 80 86 86.33 2.96 0.11

aGCC, Gouda cheese control; GCP1, Gouda cheese made with a probiotics 1 (Lb. plantarum H4) adjunct culture; GCP2, Gouda cheese made witha probiotics 2 (Lb. fermentum H9) adjunct culture.

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probiotics as adjunct cultures for manufacturing Gouda cheeseinfluenced remarkable changes in the microbial communityand the metabolism of various chemical compositions duringripening.

Peptide Profiling of Gouda Cheese. The peptidesgenerated from Gouda cheese were identified by directMALDI-TOF/MS/MS in the m/z range from 500 to 4500 Da.As shown in Table 5, 30 peptide fragments were derived fromαs1-casein (17), αS2-casein (2), β-casein (10), and κ-casein (1).The peptide profiles of cheese samples differed by starter culturesused for the manufacture of Gouda cheese. For example, theeight fragments of LPQYLKT from the center of αS2-casein,HPIKHQGLPQE from the center of αS1-casein, RPKHPIKHQ-GLPQEV and RPKHPIKHQGLPQEVL from the N-terminalof αS1-casein, GPVRGPFPI, VLGPVRGPFP, YQEPVLGPVRGP,and QEPVLGPVRGPFP from the center of β-casein wereidentified only in GCP1. However, only two peptide fragments,αS1-casein-derived HPIKHQGLPQ and β-casein-derived VSK-VKEAMAPKHKEMPFPKYPVEPF, were detected in GCP2.Interestingly, LPQYLKT (αS2-casein f176−182) and VSKVKE-AMAPKHKEMPFPKYPVEPF (β-casein f95−119) detected onlyin probiotic cheeses as well as VPSERYLGY (αS1-casein f86−94)detected in GCC were newly isolated in this study.

■ DISCUSSIONSeveral studies have reported on the microbial composition,chemical and sensory properties, and ripening of cheese madewith probiotic adjunct cultures.2−4,6 However, manufacturing offunctional cheeses by addition of probiotics has not beenstudied. Therefore, in this study, we investigated the change inmicrobial diversity induced by probiotics as well as chemicalproperties such as production of organic acids and peptidesduring ripening periods. Several criteria have been put forwardfor the selection of adjunct strains of H4 and H9 for use inprobiotic Gouda cheese production. The addition of probioticsdid not affect the composition of Gouda cheese except forlactose content. All cheese samples contained traces of lactoseranging from 0.018 to 0.026%. Particularly GCP2 had thelowest amount of lactose, because most of the lactose isremoved with the whey during draining and used as a substratefor starter and probiotic microorganisms during the saltingand drying steps before ripening in cheese manufacturing.26

However, moisture, MNFS, and FDM as well as S/M in thisstudy were not significantly different among the three types ofcheese samples even after ripening for 8 weeks as reported inprevious studies.27 In addition, application of probiotics forGouda cheese did not adversely influence cheese composition.Lactose metabolism, which is influenced by probiotics, wasassessed as the change in organic acid content during ripening.The amount of lactic acid produced was much greater thanother organic acids. Lc. lactis, Lc. lactis subsp. cremoris, and Lb.plantarum, a facultative heterofermentative LAB, metabolizelactose to lactic acid.28 A higher reduction rate of lactic acid wasobserved in probiotic cheeses than in GCC at the late stages ofripening. Some LAB such as Lb. plantarum and Lb. pentosus areable to degrade lactic acid under anaerobic conditions usingcitrate as an electron acceptor.29 There was an increase in citricacid production over the 4 week ripening period, followed by adecrease in probiotic cheeses. This pattern of citric acid contentcorrelates with previous studies by McGregor and White.30

Citric acid content is influenced by the conversion to pyruvate,acetic acid, and flavor compounds such as acetaldehyde, anddiacetyl by citrate-fermenting microorganisms such as Lc. lactisT

able

4.Taxon

omic

Classification

oftheBacterial

16SrRNAGeneSequ

encesat

Genus

Level(R

elativeAbu

ndance

>0.01%)of

Gou

daCheesedu

ring

theRipeningPeriod

GCCa

GCP1

GCP2

phylum

order

family

genus

0weeks

2weeks

4weeks

6weeks

8weeks

0weeks

2weeks

4weeks

6weeks

8weeks

0weeks

2weeks

4weeks

6weeks

8weeks

Firm

icutes

Lactobacillales

Streptococcaceae

Lactococcus

99.69

99.79

99.56

99.18

99.08

99.12

98.45

96.45

96.67

96.28

97.73

96.09

96.54

96.60

96.71

Firm

icutes

Lactobacillales

Streptococcaceae

Streptococcus

0.039

0.083

0.047

0.116

0.038

0.163

0.083

0.401

0.272

0.325

0.265

0.298

0.438

0.330

0.209

Firm

icutes

Lactobacillales

Streptococcaceae

unclassified

0.013

Firm

icutes

Lactobacillales

Leuconostocaceae

Leuconostoc

0.28

0.13

0.39

0.69

0.88

0.36

0.88

1.75

1.51

1.55

0.58

1.28

0.96

0.85

0.79

Firm

icutes

Lactobacillales

Leuconostocaceae

unclassified

0.012

0.015

0.014

Firm

icutes

Lactobacillales

Lactobacillaceae

Lactobacillus

0.35

0.58

1.32

1.49

1.79

1.40

2.28

1.92

2.16

1.56

Firm

icutes

Lactobacillales

Lactobacillaceae

unclassified

0.057

0.045

0.017

0.030

0.014

0.041

0.014

Firm

icutes

Lactobacillales

unclassified

unclassified

0.013

0.028

Proteobacteria

Xanthom

onadales

Xanthom

onadaceae

Stenotrophom

onas

0.014

aGCC,G

ouda

cheese

control;GCP1

,Gouda

cheese

madewith

aprobiotics1(Lb.plantarum

H4)

adjunctculture;GCP2

,Gouda

cheese

madewith

aprobiotics2(Lb.ferm

entum

H9)

adjunctculture.

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subsp. lactis and Leuconostoc.28,31,32 Additionally, citric acid actsas a substrate and product in the Krebs or citric acid cycle.33

Acetic acid is produced from citric acid, lactose, and aminoacids.34 The concentration of acetic acid decreased withripening time, although its level in cheeses fluctuated. Thedisappearance rate of acetic acid in GCP2 was significantlyhigher than in other cheeses. However, the irregular changesin acetic acid correlate with the findings of other studies,33 andthis could be related to the role of acetic acid as an intermediatein biochemical pathways. A continuous increase in pyruvic acidthrough ripening was observed. Pyruvic acid is produced fromcitric acid by starter cultures and acts as a key intermediate insugar metabolism as well as a substrate for several metabolicreactions.35 A small amount of propionic acid was detected onlyin probiotic Gouda cheese. Ocando et al.36 reported that pro-pionic acid production in cheeses was probably due to NSLAB,

and Lactobacillus produced propionic acid in cheese duringripening. Formic acid was also produced only in probioticcheeses. GCP1 produced the highest amount of formic acidin the late stages of ripening. This might be explained by thehigher amount of Streptococcaceae in probiotic cheeses, thegrowth of which is promoted by Lactobacillus, which producesformic acid from lactose.37 Formic acid was formed and increasedduring the initial period of ripening and then remained constantin pickled white cheese33 and Mozzarella cheese.38

The microbial community was changed by probiotics supple-mentation, which led to higher microbial diversity, richness, andevenness based on the Chao 1, Ace, and Shannon estimators.Indeed, more diverse species were identified in probiotic cheesesthan in GCC. Lb. plantarum and Lb. fermentum as adjunctcultures were detected only in GCP1 and GCP2, respectively. Inparticular, the proportion of Lb. plantarum gradually increased

Figure 1. Taxonomic classification of (A) total bacteria; (B) SLAB bacterial reads at species level retrieved from pooled DNA amplicons from Goudacheese made with a probiotics adjunct culture during the ripening period.

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and reached the highest level of 1.76% among NSLAB. In aprevious study,39 Lb. plantarum concentration was maintainedafter cheese ripening; however, the growth of Lb. fermentumwas severely inhibited during ripening, as the bacteria wassensitive to salt (5−6.5%) and low ripening temperatures below10 °C. Moreover, Lb. plantarum is known as one of the mostcommon and dominant NSLAB species found in cheese.40,41

Several authors reported that the manufacture of cheese in openvats could result in wild LAB from milk, which can grow andreach high numbers in cheese during ripening.42 Furthermore,the use of adjunct cultures that increase the growth of otherLAB, particularly Lactobacillus, during ripening in Manchegocheese4 and Cheddar cheese42 has been documented. However,lactobacilli, even the group of family Lactobacillaceae, were not

Figure 2. Taxonomic classification of (A) NSLAB bacterial reads at species level retrieved from pooled DNA amplicons; (B) proportion of adjunctprobiotics from Gouda cheese made with a probiotics adjunct culture during the ripening period.

Figure 3. Heat map showing the relative abundances and distribution of representative 16S rRNA gene tag sequences classified at species level. Thecolor code indicates differences in the relative abundance from the mean, ranging from red (negative) through black (the mean) to green (positive).

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completely detected in GCC in this study. The proportion ofLn. pseudomesenteroides in GCC increased with ripening, andthe species became abundant among the subdominant groupinstead of the various species present in probiotic cheeses. Thespecies showed high tolerance toward acidic environments andgreat proteolytic activity,43 which indicates that the number of

peptides released from GCC was similar to that of probioticcheeses.H4 caused noticeable modifications in the peptide profiles

due to enhanced secondary proteolysis by Lb. plantarum. Atotal of eight peptide fragments were detected only in GCP1compared to GCC. However, addition of H9 released two more

Figure 4. Canonical correspondence analysis (CCA) ordination diagram of bacterial communities associated with (A) chemical compositions and(B) organic acid.

Table 5. Peptide Profile of Gouda Cheese Derived from α-, β-, and κ-Caseins

m/z protein position sequence GCCa GCP1 GCP2

826 β-casein 199−206 GPVRGPFP ● ● ●851 κ-casein 96−102 ARHPHPH ● ● ●862 αS2-casein 176−182 LPQYLKT ●875 αS1-casein 1−6 RPKHPIK ● ● ●896 αS2-casein 191−197 KPWIQPK ● ● ●920 αS1-casein 86−93 VPSERYLG ●939 β-casein 199−207 GPVRGPFPI ●991 αS1-casein 26−35 APFPEVFGK ●1001 β-casein 60−68 YPFPGPIPN ● ●1012 αS1-casein 1−8 RPKHPIKH ● ● ●1038 β-casein 197−206 VLGPVRGPFP ●1083 αS1-casein 86−94 VPSERYLGY ● ●1140 αS1-casein 1−9 RPKHPIKHQ ● ● ●1154 αS1-casein 4−13 HPIKHQGLPQ ●1197 αS1-casein 1−10 RPKHPIKHQG ●1248 αS1-casein 26−36 APFPEVFGKEK ●1264 β-casein 195−206 EPVLGPVRGPFP ● ● ●1283 αS1-casein 4−14 HPIKHQGLPQE ●1311 β-casein 193−204 YQEPVLGPVRGP ●1392 β-casein 194−206 QEPVLGPVRGPFP ●1407 αS1-casein 1−12 RPKHPIKHQGLP ● ● ●1535 αS1-casein 1−13 RPKHPIKHQGLPQ ● ● ●1589 β-casein 195−209 EPVLGPVRGPF PIIV ●1664 αS1-casein 1−14 RPKHPIKHQGLPQE ● ● ●1763 αS1-casein 1−15 RPKHPIKHQGLPQEV ●1876 αS1-casein 1−16 RPKHPIKHQGLPQEVL ●1880 β-casein 193−209 YQEPVLGPVRGPFPIIV ● ● ●1990 αS1-casein 1−17 RPKHPIKHQGLPQEVLN ● ● ●2763 αS1-casein 1−23 RPKHPIKHQGLPQEVLNENLLRF ●2914 β-casein 95−119 VSKVKEAMAPKHKEMPFPKYPVEPF ●

aGCC, Gouda cheese control; GCP1, Gouda cheese made with a probiotics 1 (Lb. plantarum H4) adjunct culture; GCP2, Gouda cheese made witha probiotics 2 (Lb. fermentum H9) adjunct culture.

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peptide fragments from cheese proteins than GCC. The smallpeptides were released metabolically by the microflora incheese as well as rennet, plasmin, and cell envelope proteinasesfrom milk proteins.44 Moreover, the coagulant and enzymesfrom SLAB and NSLAB of the cheese subsequently degradecasein to peptide.45 The supplementation of probiotics mayparticipate in the metabolism of cheese proteins such as α-, β-,and κ-caseins. The peptides specifically released from GCP1and GCP2 have been reported to have various bioactivitiessuch as antimicrobial, ACE inhibitory, antioxidant, antiobesity,and antihypertensive effects.45−50 The newly isolated peptidesin this study, LPQYLKT, VPSERYLGY, and VSKVKEAMA-PKHKEMPFPKYPVEPF, are expected to have health-promotingeffects, because the selected strains, H4 and H9, have beenshown to enhance antioxidant activity and/or reduce micellarcholesterol solubility in the form of fermented milk protein.13,14

However, further studies are needed to fully understand theirbioactivities.In conclusion, we suggest that adjunct cultures of lactobacilli

have only a relatively minor influence on the chemical composi-tion of Gouda cheese, although the highest amounts of propionicacid in GCP2 and formic acid in GPC1 were observed at theend of ripening. The microbial community influenced by startercultures with probiotics may contribute to lactose metabolismduring ripening. The proportions of Lactobacillus, Leuconostoc,and Streptococcus were increased by addition of H4 and H9 asadjunct cultures. Specifically, the proportion of H4 increasedto 1.76% at the end of ripening. Moreover, several peptidefragments with health-promoting activities were additionallydetected in probiotic cheeses compared to GCC. Statistical CCAtriplot analysis indicated that several properties, including lactose,citric acid, formic acid, propionic acid, and lactic acid contents,were important factors for bacterial growth and communityformation. Therefore, Gouda cheese can be an effective vehiclefor the delivery of probiotic organisms. Moreover, the additionof H4 and H9 was associated with specific changes of the sub-dominant microbial group, mainly affecting specific metabolismof lactose and protein. However, optimization of the dosages ofthese probiotics as adjunct cultures during the manufacture ofprobiotic Gouda cheese should be performed.

■ AUTHOR INFORMATION

Corresponding Authors*(S. H. Kim) Phone: +82-2-3290-3055. Fax: +82-2-3290-3506.E-mail: saehkim@korea.ac.kr.*(Y. Kim) Phone: +82-63-270-2606. Fax: +82-63-270-2612.E-mail: ykeys2584@jbnu.ac.kr.

Author Contributions∥N. S. Oh and J. Y. Joung contributed equally to this study.

FundingThis research was supported by the High Value-Added FoodTechnology Development Program of the Korea Institute ofPlanning and Evaluation for Technology in Food, Agriculture,Forestry, and Fisheries (iPET), and the Ministry for Food,Agriculture, Forestry, and Fisheries of Republic of Korea(314068-03-1-HD020) and a grant from Cooperative ResearchProgram for Agriculture Science & Technology Development(Project No. PJ01088202), Rural Development Administration,Republic of Korea.

NotesThe authors declare no competing financial interest.

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