Characterization of the Microbial Diversity and Chemical Composition

Subscriber access provided by La Trobe University Library

Article

Characterization of the Microbial Diversity and Chemical Composition of Gouda Cheese made by Potential Probiotic Strains as an Adjunct Starter Culture Nam Su Oh, Jae Yeon Joung, Ji Young Lee, Sae Hun Kim, and Younghoon Kim J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b02689 • Publication Date (Web): 08 Sep 2016 Downloaded from http://pubs.acs.org on September 13, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 38

Journal of Agricultural and Food Chemistry

1

2

Characterization of the Microbial Diversity and Chemical Composition of

3

Gouda Cheese made by Potential Probiotic Strains

4

as an Adjunct Starter Culture

5

Nam Su Oha*, Jae Yeon Jounga,b*, Ji Young Leea,b, Sae Hun Kimb#, and Younghoon Kimc#

6 7

8

a

R&D Center, Seoul Dairy Cooperative, Ansan, Kyunggi 15407, South Koreaa

9

b

Department of Biotechnology, College of Life Sciences and Biotechnology, Korea University,

10

Seoul 02841, South Korea

11

c

12

Jeonju 54896, South Korea

Department of Animal Science and Institute of Milk Genomics, Chonbuk National University,

13

14

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

15

16

Corresponding author: S. H. Kim, and Y. Kim

17

Tel: +82-63-270-2606; [email protected]

18

Fax:

+82-63-270-2612;

E-mail:

19

20

1

ACS Paragon Plus Environment

[email protected]

or


21

Acknowledgement

22

This research was supported by the High Value-Added Food Technology Development

23

Program of the Korea Institute of Planning and Evaluation for Technology in Food,

24

Agriculture, Forestry, and Fisheries (iPET), and the Ministry for Food, Agriculture, Forestry,

25

and Fisheries of Republic of Korea (314068-03-1-HD020) and a grant from the Next-

26

Generation BioGreen 21 Program (Project No. PJ0118142016), Rural Development

27

Administration, Republic of Korea.

28 29

Notes

30

The authors declare no competing financial interests.

31 32

2


Page 2 of 38

Page 3 of 38


33

Abstract

34

We characterized the microbial diversity and chemical properties of Gouda cheese made

35

by probiotics during ripening periods. Lactobacillus plantarum H4 (H4) and Lactobacillus

36

fermentum H9 (H9), which demonstrate probiotic properties and bioactivity, were used as

37

adjunct starter cultures. Gouda cheese made with H4 (GCP1) and H9 (GCP2) demonstrated

38

the highest production of formic acid and propionic acid, respectively. Moreover, the

39

bacterial diversity, including richness and evenness of non-starter lactic acid bacteria

40

(NSLAB), increased in probiotic cheeses. Specifically, Lactobacillus, Leuconostoc, and

41

Streptococcaceae were present at higher concentrations in probiotic cheeses than in control

42

Gouda cheese (GCC). The proportion of H4 in GCP1 increased and culminated at 1.76%,

43

while H9 in GCP2 decreased during ripening. Peptide profiles were altered by addition of

44

probiotics and included various bioactive peptides. In particular, three peptide fragments are

45

newly detected. Therefore, Gouda cheese could be used as an effective probiotic carrier for

46

H4 and H9.

47 48 49

Keywords: Gouda cheese, Adjunct cultures, Probiotics, Microbial community, Organic acid,

50

Peptide profiling

51

3



52

Page 4 of 38

Introduction

53

Gouda cheese is produced from pasteurized milk acidified by mesophilic lactic acid

54

bacteria (LAB) such as Lactococcus lactis subsp. lactis (Lc. lactis subsp. lactis), Lc. lactis

55

subsp. cremoris, Lc. lactis subsp. lactis biovar diacetylactis, and Leuconostoc mesenteroides

56

subsp. cremoris (Ln. mesenteroides subsp. cremoris) 1. Gouda cheese is generally matured for

57

2–3 months at approximately 13 to 15°C 2. Adjunct cultures of Lactobacillus have been

58

studied to control the ripening process and the growth of the microbial composition of non-

59

starter LAB (NSLAB) in Cheddar cheese 3, Manchego cheese 4, and Gouda cheese

60

manufacturing, as well as to improve organoleptic properties. Adjunct cultures can change

61

the microbial community of NSLAB, and this coincides with cheese proteolysis and lipolysis

62

during ripening 6. Moreover, cheese provides a valuable alternative to fermented milks and

63

yogurts as a probiotic food carrier by incorporating of probiotics in cheese as adjunct cultures.

64

Probiotic food products including probiotic cheese must demonstrate their efficacy and

65

maintain the probiotic viability in the final products 7. And the incorporation of probiotics

66

should not imply a loss of quality of products 7. Beneficial effects on health related to

67

probiotic cheese have been reported that probiotic fresh cheese containing Lb. acidophilus, B.

68

bifidum, and Lb. paracasei demonstrated immune-modulating capacity in mice

69

probiotic Edam cheese contacting Lb. rhamnosus on the risk of dental caries was studied 9.

70

However, there are few studies demonstrating healthy functional Gouda cheese with probiotic

71

adjunct cultures. The use of bacterial strains with health benefits could be possible strategy

72

for the cheese industry, to address the increased demand for new or special cheeses. Next-

73

generation sequencing (NGS) methodologies provide a powerful tool to analyze complex

74

microbial

communities

of

fermented

food

materials,

4


since

multiplex

8

5

and

barcoded

Page 5 of 38


75

pyrosequencing allows the analysis of multiple samples in a single run inexpensively 10-11. A

76

previous study reported that Cheddar cheese made with probiotics strains of Lactobacillus

77

isolated from the human small intestine has been characterized for its probiotic potential. The

78

probiotic strains sustained at a high viability in cheese during up to 120 days of ripening 12. In

79

this study, Lactobacillus plantarum H4 (H4) and Lactobacillus fermentum H9 (H9), isolated

80

from infant feces, were selected as probiotic adjunct cultures. The strains exhibited probiotic

81

potential such as acid- and bile-tolerance, proteolytic activity, adhesion to intestine, and

82

additional bioactivities such as antioxidant and cholesterol lowering activities 13-14. The main

83

objective of this study was to investigate the effects of probiotics as adjunct cultures on the

84

ripening of Gouda cheese. Accordingly, the microbial communities and chemical properties

85

were analyzed.

86

5



87

88

Page 6 of 38

Materials and Methods Bacterial culture

89

Freeze-dried direct vat starter culture (DVS) Lc. lactis subsp. cremoris, Leuconostoc, Lc.

90

lactis subsp. Lactis, and Lc. lactis subsp. lactis biovar diacetylactis with code CHN-11 were

91

obtained from Chr. Hansen (Horsholm, Denmark). H4 and H9 probiotic cultures were

92

selected for this study, based on their probiotic potential and bioactivities. For initial strain

93

selection, Lactobacillus strains were isolated from plant and human feces. Briefly, the plant

94

and feces samples were weighed and homogenized for 30 s in saline and diluted. Aliquots of

95

serial dilutions were plated on de Man, Rogosa, and Sharpe (MRS) agar (Difco Laboratories,

96

MI, USA) and incubated at 37°C for 48–72 h. In total, 450 strains were isolated and

97

evaluated their probiotic potential, using various tests such as acid and bile tolerance,

98

adhesion to intestine, protein hydrolysis activity, antioxidant activity, and cholesterol-

99

reducing ability (data not shown)

13-15

. Then, we performed complete genome sequencing of

100

selected two probiotic strains and the bioproject number for Lb. plantarum H4 and Lb.

101

fermentum

102

(http://www.ncbi.nlm.nih.gov/genbank/) as accession numbers of PRJNA325681 and

103

PRJNA325680, respectively.

H9

have

been

deposited

in

the

NCBI

GenBank

104

105

Manufacture of probiotic Gouda cheese

106

Three separate trials of Gouda cheese manufacturing were performed with or without

107

probiotics. Raw milk was obtained from Seoul Dairy Cooperative (Ansan, Kyunggi, Korea)

108

to make Gouda cheese. Probiotic Gouda cheese was manufactured in a pilot scale cheese vat. 6


Page 7 of 38


109

Specifically, 50 kg of raw milk was pasteurized at 72°C for 15 s and cooled down to 34°C.

110

The pasteurized milk was inoculated with 0.016% of starter culture CHN-11 (Chr. Hansen,

111

Hørsholm, Denmark) and 0.01% of probiotics (H4 and H9, 6.35 ± 0.02 log CFU/mL and 7.22

112

± 0.02 log CFU/mL, respectively), and 0.01% of CaCl2 was added. The species which

113

compose starter culture CHN-11 were Lactococcus lactis subsp. cremoris, Leuconostoc,

114

Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. lactis biovar diacetylactis. When

115

the acidity increased by 0.01% (after 40 min, 0.1422%→0.1520%), 0.02% rennet (Chr.

116

Hansen, New Zealand) was added to induce the formation of curds. The milk was allowed to

117

coagulate for 60 min, and the coagulum was then cut into 9 mm cube-shaped particles. After

118

the curds were simultaneously stirred for 30 min, the whey was removed (30% of the volume

119

of the milk) and water (20% of the volume of the milk) at 38°C was added. The curds were

120

stirred for 20 min. Following stirring, 30% (the volume of the milk) of whey was removed

121

and 20% (the volume of the milk) of 73°C water was added (2.5 kg/5min) to the stirring

122

curds (for 20 min), as the temperature of the curd-whey mixture gradually increased from

123

34°C to 37°C. The whey was drained, and the curd was put into a mold and pre-pressed for

124

60 min. The curd was pressed (for 12 h) until the pH reached 5.05–5.10. The probiotic Gouda

125

cheese was removed from the mold and soaked in a 20% saturated brine solution at 5°C for

126

24h. The cheese was allowed to dry for 2 days. Control Gouda cheese was manufactured by

127

the same manufacturing process as probiotic cheese but without supplementation of

128

probiotics. The Gouda cheese was packed in cheese wax and ripened for 8 weeks at 15°C.

129

Samples of Gouda cheese were collected for analysis every 2 weeks post manufacture.

130

131

Chemical compositions of Gouda cheese 7



132

Page 8 of 38

Gouda cheeses were grated and analyzed in triplet for protein, fat, lactose, moisture, pH,

133

and salt in moisture (S/M), fat in dry matter (FDM), and moisture in nonfat substrates (MNFS)

134

as described by the association of official analytical chemists chapter 33.2 and 33.7

135

Organic acid was analyzed with HPLC-UVD as the method of Ong et al.

136

modification which the concentration of sulfuric acid used as mobile phase and extraction

137

buffer was changed from 0.009N to 0.005N.

17

16

.

with slight

138

139

PCR amplification and pyrosequencing

140

PCR amplification was performed using primers targeting from V1 to V3 regions of the

141

16S rRNA gene with extracted DNA from fifteen different cheese samples per the method of

142

Riquelme

143

CCTATCCCCTGTGTGCCTTGGCAGTC-TCAG-AC-GAGTTTGATCMTGGCTCAG-3’ ;

144

underlining

145

CCATCTCATCCCTGCGTGTCTCCGAC-TCAG-X-AC-WTTACCGCGGCTGCTGG-3’;

146

‘X’ indicates the unique barcode for each subject) (http://oklbb.ezbiocloud.net/content/1001).

147

The amplifications was carried out under the following conditions: initial denaturation at

148

95 °C for 5 min, followed by 30 cycles of denaturation at 95 °C for 30 sec, primer annealing

149

at 55 °C for 30 sec, and extension at 72 °C for 30 sec, with a final elongation at 72 °C for 5

150

min. The PCR product was confirmed by using 2% agarose gel electrophoresis and visualized

151

under a Gel Doc system (BioRad, Hercules, CA, USA). The amplified products were purified

152

with the QIAquick PCR purification kit (Qiagen, Valencia, CA, USA). Equal concentrations

153

of purified products were pooled together and removed short fragments (non-target products)

154

with Ampure beads kit (Agencourt Bioscience, MA, USA). The quality and product size were

10

.

For

sequence

bacterial

indicates

amplification,

the

target

barcoded

region

8


primers

primer)

of

and

27F

518R

5’-

5’-

Page 9 of 38


155

assessed on a Bioanalyzer 2100 (Agilent, Palo Alto, CA, USA) using a DNA 7500 chip.

156

Mixed amplicons were conducted emulsion PCR, and then deposited on Picotiter plates. The

157

sequencing was carried out at Chunlab, Inc. (Seoul, Korea), with GS Junior Sequencing

158

system (Roche,Branford, CT, USA) according to the manufacturer’s instructions.

159 160

Pyrosequencing data analysis

161

The basic analysis was conducted according to the previous descriptions in other studies

162

18-20

163

product. The sequences of the barcode, linker, and primers were removed from the original

164

sequencing reads. Any reads containing two or more ambiguous nucleotides, low quality

165

score (average score < 25), or reads shorter than 300bp, were discarded. Potential chimera

166

sequences were detected by the bellerophone method, which is comparing the BLASTN

167

search results between forward half and reverse half sequences

168

sequences, the taxonomic classification of each read was assigned against the EzTaxon-e

169

database (http://eztaxon-e.ezbiocloud.net)

170

type strains that have valid published names and representative species level phylotypes of

171

either cultured or uncultured entries in the GenBank database with complete hierarchical

172

taxonomic classification from the phylum to the species. The richness and diversity of

173

samples were determined by Chao1 estimation and Shannon diversity index at the 3%

174

distance. Random subsampling was conducted to equalize read size of samples for comparing

175

different read sizes among samples. The pyrosequencing data was analyzed using the

176

CLcommunity program (Chunlab Inc., Seoul, Korea).

. Obtained reads from the different samples were sorted by unique barcodes of each PCR

21

. After removing chimera

22

, which contains 16S rRNA gene sequence of

177 9



178

Peptide profiling by MALDI-TOF/MS/MS

179

The water soluble nitrogen (WSN) of cheese samples were prepared with the method of 23

180

Ardö and Polychroniadou

181

times and freeze dried. The cheese peptide analysis was performed by the method of Oh et al.

182

24

with slight modification. WSN was extracted from cheese four

with MALDI-TOF/MS using Bruker Autoflex (Bruker Daltonics, Bremen, Germany).

183 184

Statistical analysis

185

All data were expressed as means ± SD. Statistical significance for the differences

186

between the groups was assessed using Duncan’s multiple range tests. IBM SPSS statistics

187

software version 22 (IBM Corp., Armonk, NY, USA) was used to perform all statistical tests.

188

Values of P < 0.05 were considered to indicate a significant difference.

189

190

10


Page 10 of 38

Page 11 of 38


191

192

Results Chemical composition of Gouda cheese

193

The chemical composition of Gouda cheese is shown in Table 1. No significant

194

difference in the chemical compositions such as the contents of protein, fat, moisture, and

195

S/M of three types of Gouda cheese was observed. However, the amount of lactose was the

196

lowest in Gouda cheese made with H9 (GCP2) at the initial stages of ripening, but reduced

197

entirely in all cheese samples. The moisture content was also significantly reduced after

198

ripening, with a slight increase in the protein, fat, and S/M content of the cheeses. The lactose

199

metabolic activity of the microorganisms in Gouda cheese was assessed by estimating the

200

production of organic acids such as citric acid, pyruvic acid, lactic acid, acetic acid, propionic

201

acid, and formic acid (Table 2). The concentration of organic acids was significantly affected

202

by the addition of probiotics and ripening time. The main organic acids in Gouda cheese

203

throughout ripening were citric, lactic, and acetic acids. An increase in concentration during

204

ripening was observed in pyruvic and propionic acids, whereas the levels of lactic and acetic

205

acids were irregular, demonstrating a slight decreasing tendency. The concentration of citric

206

acid increased in the early stages of ripening and eventually decreased. In addition, the

207

disappearance rate of citric, lactic, and acetic acids in probiotic cheeses was significantly

208

higher than for GCC. Propionic and formic acids were hardly detected in GCC, and the level

209

of propionic acid exponentially increased in Gouda cheese made with H4 (GCP1) and GCP2

210

at the 6-week and 4-week ripening period, respectively. The level of formic acid in GCP2

211

was the highest at the beginning of ripening (week 0), but decreased during the ripening

212

period. However, it gradually increased in GCP1 for 6 weeks of ripening, with the highest

213

levels observed at the end of the ripening period. 11



214 215

Comparison of species and diversity estimates of bacterial community in Gouda cheese

216

Samples of Gouda cheese made with probiotics collected at 0, 2, 4, 6, and 8 weeks of

217

ripening were analyzed for microbial diversity by high-throughput sequencing (Table 3). A

218

total of 110,679 bacterial sequencing reads with an average sequence length of 397.94bp and

219

an average of 7,379 sequencing reads per sample were obtained from three types of Gouda

220

cheese. The Good’s coverage index, an estimator of sampling completeness, for each data set

221

was above 99% in all samples, indicating that the rarified sequencing depth was sufficient to

222

evaluate the bacterial diversity of Gouda cheese 25. The rarefaction curves (data not shown)

223

were approximately an asymptote, which indicated that the analyzed data sufficiently

224

reflected the bacterial community and the entire bacterial diversity of cheese samples. The

225

alpha diversity analysis revealed that Chao 1 and Ace estimators, which provides the richness

226

of bacterial species in a sample, decreased during the first 2 weeks of ripening and increased

227

until week 6, then decreased again until the end of ripening in all cheese samples. In

228

particular, GCP1 and GCP2 made with probiotics showed higher values of bacterial richness

229

parameters than GCC. Moreover, the Shannon’s diversity index revealed higher diversity in

230

probiotic cheeses, which indicated that the evenness of bacterial species distribution in GCP1

231

and GCP2 was higher than in GCC.

232

233

Bacterial communities of Gouda cheese

234

The 16S rRNA gene sequencing reads were classified into different taxonomies. The

235

relative abundance of each sample was generated into phylum, order, family, genus, and 12


Page 12 of 38

Page 13 of 38


236

species levels (Table 4, Figures 1 and 2). At the phylum level, Firmicutes dominated, and

237

Proteobacteria was identified only in GCP2 at week 8 of ripening. At the family level, all

238

cheese samples were dominated by the family Streptococcaceae (> 96.4%), however, the

239

second most abundant family varied among samples. As shown in Table 4, Leuconostocaceae

240

was the only family group (except Streptococcaceae) present in GCC, with an increase in

241

proportion from 0.28% to 0.88% during ripening. GCP1 contained Leuconostocaceae (1.21%)

242

and Lactobacillaceae (1.13%) as the second dominant family with similar proportion. The

243

proportion of Leuconostocaceae increased until week 4 of ripening and then decreased,

244

whereas Lactobacillaceae increased through the ripening period from 0.35% to 1.81%. The

245

Lactobacillaceae family was the second most abundant group, of which the proportion was

246

approximately two-fold higher than that of Leuconostocaceae in GCP2. Notably,

247

Xanthomonadaceae, which belongs to the phylum Proteobacteria was detected only in GCP2.

248

Likewise, further classification to genus level indicated that bacterial communities varied

249

considerably among the three types of cheese samples during ripening. Specifically, probiotic

250

cheeses showed more diverse bacterial community than GCC.

251

Pyrosequencing revealed that five genera (relative abundance > 0.01%) of Lactococcus,

252

Streptococcus, Leuconostoc, Lactobacillus, and Stenotrophomonas were identified in Gouda

253

cheese. The most abundant genus was Lactococcus, with over 96.1% abundance in all cheese

254

samples. The second dominant genus differed by the type of starter culture used for the

255

manufacture of Gouda cheese: Leuconostoc for GCC, Leuconostoc and Lactobacillus for

256

GCP1, and Lactobacillus for GCP2. Lactobacillus, unclassified Lactobacillaceae, and

257

Stenotrophomonas were detected only in probiotic cheeses. A sequence that could not be

258

assigned taxonomic affiliations at a 97% level of similarity was labeled as “unclassified”.

13



Page 14 of 38

259

The taxonomic classification of cheese bacteria at species level is presented, dividing

260

into SLAB and NSLAB in Figures 1 and 2, respectively. Lc. lactis subsp. cremoris and Lc.

261

lactis groups comprised most (94.3% ~ 99.9%) of the microbial community in Gouda cheese.

262

In particular, Lc. lactis subsp. cremoris tended to decrease with ripening, in GCP1 and GCP2.

263

Figure 2 shows the dynamic and diverse NSLAB bacterial community. In GCC, the

264

proportion of Lactococcus decreased, whereas that of Ln. pseudomesenteroides increased, as

265

ripening

266

pseudomesenteroides proportions compared to GCC, except that Lb. plantarum was

267

specifically detected, and the proportion increased throughout ripening from 0.34% to 1.76%.

268

Lb. fermentum was identified only in GCP2, although the proportion dramatically increased

269

initially and then gradually decreased until week 8 of ripening. Unclassified Lactobacillus,

270

Lactobacillales, and Lactobacillaceae were present only in probiotic cheeses. Moreover,

271

unclassified Lactococcus and unclassified Streptococcaceae were present at higher

272

proportions in GCP1 and GCP2 than in GCC.

progressed.

GCP1

showed

a

similar trend for Lactococcus

and

Ln.

273

The results of the microbial community and diversity analyses based on taxonomy

274

classification at species level corresponded with the results of the heat map analysis of

275

microbial communities in cheese samples, which were distributed based on the main six

276

species (Figure 3). Each cheese sample, based on the extent of ripening, contained a sample-

277

specific bacterial community. The unweighted pair group method with arithmetic mean

278

(UPGMA) tree constructed on the basis of the dissimilarity of the bacterial community in

279

cheese samples indicated two clusters as group 1 (GCP1 6 week, GCP1 4 week, GCP2 6

280

week, GCP2 8 week, GCP2 4 week, GCP1 8 week, GCP2 2 week, and GCP2 0 week) and

281

group 2 (GCC 8 week, GCC 4 week, GCC 6 week, GCP1 2 week, GCC 2 week, GCC 0 week,

14


Page 15 of 38


282

and GCP1 0 week). Cheese samples manufactured from the same starter culture clustered

283

together and samples with similar ripening degree clustered closely.

284

285

Statistical comparison of the microbial community structure of Gouda cheese

286

Canonical correspondence analysis (CCA) results for several microbial assemblages in

287

relation to chemical compositions of Gouda cheese and organic acids are shown in Figure 4.

288

The A and B triplot shows the relationship between the type of starter culture, bacterial

289

composition, and chemical composition (protein, fat, lactose, moisture, S/M, FDM, MNFS,

290

and organic acids). The CCA triplot indicated that several properties, including lactose, citric

291

acid, formic acid, propionic acid, and lactic acid contents were important for bacterial growth

292

and community formation. These factors also had an effect on the distribution of Gouda

293

cheese clusters according to probiotics and cheese ripening. In contrast, the chemical

294

compositions analysis (Table 1), which is the CCA triplot for chemical compositions showed

295

no discernible trend. However, the triplot analysis for organic acid showed that formic acid

296

and propionic acid were considered the key factors for the distribution of cheese samples

297

according to ripening period. Lactobacillus, Leuconostoc, and Streptococcaceae highly

298

correlated with formic, pyruvic, and propionic acid content. The cheese samples were

299

distributed in three groups by probiotics; particularly probiotic cheeses at initial and late

300

ripening were clustered separately. This indicates that probiotics as adjunct cultures for

301

manufacturing Gouda cheese influenced remarkable changes in the microbial community,

302

and the metabolism of various chemical compositions during ripening.

303

15



304

Page 16 of 38

Peptide profiling of Gouda cheese

305

The peptides generated from Gouda cheese were identified by direct MALDI-

306

TOF/MS/MS in the m/z range from 500 to 4,500 Da. As shown in Table 4, thirty peptide

307

fragments were derived from αs1-casein (17), αs2-casein (2), β-casein (10), and κ-casein (1).

308

The peptide profiles of cheese samples differed by starter cultures used for the manufacture

309

of Gouda cheese. For example, the eight fragments of LPQYLKT from the center of αs2-

310

casein, HPIKHQGLPQE from the center of αs1-casein, RPKHPIKHQGLPQEV and

311

RPKHPIKHQGLPQEVL from the N-terminal of αs1-casein, GPVRGPFPI, VLGPVRGPFP,

312

YQEPVLGPVRGP, and QEPVLGPVRGPFP from the center of β-casein were only identified

313

in GCP1. However, only two peptide fragments, αs1-casein derived HPIKHQGLPQ and β-

314

casein

315

Interestingly, LPQYLKT (αs2-casein f176-182) and VSKVKEAMAPKHKEMPFPKYPVEPF

316

(β-casein f95-119) detected only in probiotic cheeses as well as VPSERYLGY (αs1-casein

317

f86-94) detected in GCC were newly isolated in this study.

derived

VSKVKEAMAPKHKEMPFPKYPVEPF,

318

16


were

detected

in

GCP2.

Page 17 of 38

319

320


Discussion Several studies have reported on the microbial composition, chemical and sensory 2-4, 6

321

properties, and ripening of cheese made with probiotic adjunct cultures

322

manufacturing of functional cheeses by addition of probiotics has not been studied. Therefore,

323

in this study, we investigated the change in microbial diversity induced by probiotics as well

324

as chemical properties such as production of organic acids and peptides during ripening

325

periods. Several criteria have been put forward for the selection of adjunct strains of H4 and

326

H9, for use in probiotic Gouda cheese production. The addition of probiotics did not affect

327

the composition of Gouda cheese except for lactose content. All cheese samples contained

328

traces of lactose ranging from 0.018% to 0.026%. Particularly GCP2 had the lowest amount

329

of lactose, since most of the lactose is removed with the whey during draining, and used as a

330

substrate for starter and probiotic microorganisms during the salting and drying steps before

331

ripening in cheese manufacturing 26. However, moisture, MNFS, and FDM as well as S/M in

332

this study were not significantly different among the three types of cheese samples even after

333

ripening for 8 weeks as reported in previous studies 27. In addition, application of probiotics

334

for Gouda cheese did not adversely influence cheese composition. Lactose metabolism,

335

which is influenced by probiotics, was assessed as the change in organic acid content during

336

ripening. The amount of lactic acid produced was much greater than other organic acids. Lc.

337

lactis, Lc. lactis subsp. cremoris, and Lb. plantarum, a facultative heterofermentative LAB,

338

metabolizes lactose to lactic acid 28. The higher reduction rate of lactic acid was observed in

339

probiotic cheeses than in GCC at the late stages of ripening. Some LAB such as Lb.

340

plantarum and Lb. pentosus are able to degrade lactic acid under anaerobic conditions using

341

citrate as an electron acceptor 29. There was an increase in citric acid production over the 4 17


. However,


Page 18 of 38

342

week ripening period, followed by a decrease in probiotic cheeses. This pattern of citric acid

343

content correlates with previous studies by McGregor and White

344

influenced by the conversion to pyruvate, acetic acid, and flavor compounds such as

345

acetaldehyde, and diacetyl by citrate–fermenting microorganisms such as Lc. lactis subsp.

346

lactis and Leuconostoc

347

Krebs or citric acid cycle 33. Acetic acid is produced from citric acid, lactose, and amino acids

348

34

349

fluctuated. The disappearance rate of acetic acid in GCP2 was significantly higher than in

350

other cheeses. However, the irregular changes in acetic acid correlates with the findings of

351

other studies

352

biochemical pathways. A continuous increase in pyruvic acid through ripening was observed.

353

Pyruvic acid is produced from citric acid by starter cultures and acts as a key intermediate in

354

sugar metabolism as well as a substrate for several metabolic reactions 35. A small amount of

355

propionic acid was detected only in probiotic Gouda cheese. Ocando et al.

356

propionic acid production in cheeses was probably due to NSLAB, and Lactobacillus

357

produced propionic acid in cheese during ripening. Formic acid was also produced only in

358

probiotic cheeses. GCP1 produced the highest amount of formic acid in the late stages of

359

ripening. This might be explained by the higher amount of Streptococcaceae in probiotic

360

cheeses, the growth of which is promoted by Lactobacillus, which produces formic acid from

361

lactose

362

then remained constant in pickled white cheese 33 and Mozzarella cheese 38.

30

. Citric acid content is

28, 31-32

. Additionally, citric acid acts as a substrate and product in the

. The concentration of acetic acid decreased with ripening time, although its level in cheeses

33

, and this could be related to the role of acetic acid as an intermediate in

36

reported that

37

. Formic acid was formed and increased during the initial period of ripening and

363

The microbial community was changed by probiotics supplementation, which led to

364

higher microbial diversity, richness, and evenness based on the Chao 1, Ace, and Shannon

18


Page 19 of 38


365

estimators. Indeed, more diverse species were identified in probiotic cheeses than in GCC. Lb.

366

plantarum and Lb. fermentum as adjunct cultures were detected only in GCP1 and GCP2,

367

respectively. In particular, the proportion of Lb. plantarum gradually increased and reached

368

the highest level of 1.76% among NSLAB. In a previous study

369

concentration was maintained after cheese ripening, however the growth of Lb. fermentum

370

was severely inhibited during ripening, as the bacteria was sensitive to salt (5-6.5%) and low

371

ripening temperature below 10ºC. Moreover, Lb. plantarum is known as one of the most

372

common and dominant NSLAB species found in cheese

373

the manufacture of cheese in open vats could result in wild LAB from milk, which can grow

374

and reach high numbers in cheese during ripening 42. Furthermore, the use of adjunct cultures

375

that increase the growth of other LAB, particularly Lactobacillus, during ripening in

376

Manchego cheese

377

even the group of family Lactobacillaceae, were not completely detected in GCC in this

378

study. The proportion of Ln. pseudomesenteroides in GCC increased with ripening and the

379

species became abundant among the subdominant group instead of the various species

380

present in probiotic cheeses. The species showed high tolerance toward acidic environments

381

and great proteolytic activity

382

GCC was similar to that of probiotic cheeses.

4

and Cheddar cheese

42

39

, Lb. plantarum

40-41

. Several authors reported that

have been documented. However, lactobacilli,

43

, which indicates that the number of peptides released from

383

H4 caused noticeable modifications in the peptide profiles due to enhanced secondary

384

proteolysis by Lb. plantarum. A total of eight peptide fragments were detected only in GCP1

385

compared to GCC. However, addition of H9 released two more peptide fragments from

386

cheese proteins than GCC. The small peptides were released metabolically by the microflora

387

in cheese as well as rennet, plasmin, and cell envelope proteinases from milk proteins

19


44

.


Page 20 of 38

388

Moreover, the coagulant and enzymes from SLAB and NSLAB of the cheese subsequently

389

degrade casein to peptide

390

metabolism of cheese proteins such as α-, β-, and κ-casein. The peptides specifically released

391

from GCP1 and GCP2 have been reported to have various bioactivities such as antimicrobial,

392

ACE inhibitory, antioxidant, anti-obesity, and anti-hypertensive effects

393

isolated

394

VSKVKEAMAPKHKEMPFPKYPVEPF are expected to have health promoting effects,

395

since the selected strains, H4 and H9 have been shown to enhance antioxidant activity and/or

396

reduce micellar cholesterol solubility in the form of fermented milk protein

397

further studies are needed to fully understand their bioactivities.

peptides

in

45

. The supplementation of probiotics may participate in the

this

study,

LPQYLKT,

45-50

. The newly

VPSERYLGY,

and

13-14

. However,

398

In conclusion, we suggest that adjunct cultures of lactobacilli have only a relatively

399

minor influence on the chemical composition of Gouda cheese, although the highest amounts

400

of propionic acid in GCP2 and formic acid in GPC1 were observed at the end of ripening.

401

The microbial community influenced by starter cultures with probiotics may contribute to

402

lactose metabolism during ripening. The proportions of Lactobacillus, Leuconostoc, and

403

Streptococcus were increased by addition of H4 and H9 as adjunct cultures. Specifically, the

404

proportion of H4 increased to 1.76% at the end of ripening. Moreover, several peptide

405

fragments with health promoting activities were additionally detected in probiotic cheeses

406

compared to GCC. Statistical CCA triplot analysis indicated that several properties, including

407

lactose, citric acid, formic acid, propionic acid, and lactic acid contents were important

408

factors for bacterial growth and community formation. Therefore, Gouda cheese can be an

409

effective vehicle for delivery of probiotic organisms. Moreover the addition of H4 and H9

410

was associated with specific change of the subdominant microbial group, mainly affecting

20


Page 21 of 38


411

specific metabolism of lactose and protein. However, optimization of the dosages of these

412

probiotics as adjunct cultures during the manufacturing of probiotic Gouda cheese should be

413

performed.

414

21



415

References

416

1. Van den Berg, G.; Meijer, W. C.; Düsterhöft, E.-M.; Smit, G., Gouda and related cheeses.

417 418

Elsevier Academic Press.: London, UK, 2004. 2.

Ruyssen, T.; Janssens, M.; Van Gasse, B.; Van Laere, D.; Van der Eecken, N.; De

419

Meerleer, M.; Vermeiren, L.; Van Hoorde, K.; Martins, J. C.; Uyttendaele, M.; De Vuyst,

420

L., Characterisation of Gouda cheeses based on sensory, analytical and high-field 1H

421

nuclear magnetic resonance spectroscopy determinations: Effect of adjunct cultures and

422

brine composition on sodium-reduced Gouda cheese. International Dairy Journal 2013,

423

33 (2), 142-152.

424

3.

Milesi, M. M.; McSweeney, P. L. H.; Hynes, E. R., Viability and contribution to

425

proteolysis of an adjunct culture of Lactobacillus plantarum in two model cheese

426

systems: Cheddar cheese-type and soft-cheese type. Journal of Applied Microbiology

427

2008, 105 (2008), 884–892.

428

4.

Poveda, J. M.; Sousa, M. J.; Cabezas, L.; McSweeney, P. L. H., Preliminary observations

429

on proteolysis in Manchego cheese made with a defined-strain starter culture and adjunct

430

starter (Lactobacillus plantarum) or a commercial starter. International Dairy Journal

431

2003, 13, 169-178.

432

5.

Van Hoorde, K.; Van Leuven, I.; Dirinck, P.; Heyndrickx, M.; Coudijzer, K.; Vandamme,

433

P.; Huys, G., Selection, application and monitoring of Lactobacillus paracasei strains as

434

adjunct cultures in the production of Gouda-type cheeses. International Journal of Food

435

Microbiology 2010, 144 (2), 226-235.

436

6.

Gobbetti, M.; De Angelis, M.; Di Cagno, R.; Mancini, L.; Fox, P. F., Pros and cons for

437

using non-starter lactic acid bacteria (NSLAB) as secondary/adjunct starters for cheese

438

ripening. Trends in Food Science & Technology 2015, 45 (2), 167-178.

439

7.

da Cruz, A. G.; Buriti, F. C. A.; de Souza, C. H. B.; Fariaa, J. A. F.; Saad, S. M. I.,

440

Probiotic cheese: health benefits, technological and stability aspects. Trends in Food

441

Science & Technology 2009, 20, 344-354. 22


Page 22 of 38

Page 23 of 38

442


8.

443 444

Medici, M.; Vinderola, C. G.; Perdigo´n, G., Gut mucosal immunomodulation by probiotic fresh cheese. International Dairy Journal 2004, 14 (7), 611-618.

9.

Ahola, A. J.; Knnuttila-Yli, H.; Suomalainen, T.; Poussa, T.; Ahlsrom, A.; Meurman, J.

445

H.; Korpela, R., Short-term consumption of probiotic containing cheese and its effect on

446

dental caries risk factors. Archives of Oral Biology 2002, 47 (11), 799-804.

447

10. Riquelme, C.; Câmara, S.; Dapkevicius, M. L.; Vinuesa, P.; da Silva C. C.; Malcata, F.

448

X.; Rego, O. A., Characterization of the bacterial biodiversity in Pico cheese (an

449

artisanal Azorean food). Int J Food Microbiol. 2014, 2 (192), 86-94.

450 451

11. Solieri, L.; Dakal, T. C.; Giudici, P., Next-generation sequencing and its potential impact on food microbial genomics. Annals of Microbiology 2013, 63 (1), 21-37.

452

12. Desfosses-Foucault, E.; Dussault-Lepage, V.; Le Boucher, C.; Savard, P.; La Pointe, G.;

453

Roy, D., Assessment of probiotic viability during Cheddar cheese manufacture and

454

ripening using propidium monoazide-PCR quantification. Frotiers in Microbiology 2012,

455

3, 1-11.

456

13. Oh, N. S.; Kwon, H. S.; Lee, H. A.; Joung, J. Y.; Lee, J. Y.; Lee, K. B.; Shin, Y. K.; Baick,

457

S. C.; Park, M. R.; Kim, Y.; Lee, K. W.; Kim, S. H., Preventive effect of fermented

458

Maillard reaction products from milk proteins in cardiovascular health. Journal of Dairy

459

Science 2014, 97, 3300-3313.

460

14. Oh, N. S.; Park, M. R.; Lee, K. W.; Kim, S. H.; Kim, Y., Dietary Maillard reaction

461

products and their fermented products reduce cardiovascular risk in an animal model.

462

Journal of Dairy Science 2015, 98 (8), 5102-5112.

463

15. Argyri, A. A.; Zoumpopoulou, G.; Karatzas, K.-A. G.; Tsakalidou, E.; Nychas, G. J. E.;

464

Panagou, E. Z.; Tassou, C. C., Selection of potential probiotic lactic acid bacteria from

465

fermented olives by in vitro tests. Food Microbiology 2013, 33 (2), 282-291.

466 467 468

16. AOAC, Official methods of analysis. Association of Official Analytical Chemists: Washington, DC, USA, 2005. 17. Ong, L.; Henriksson, A.; Shah, N. P., Development of probiotic Cheddar cheese 23



469

containing Lactobacillus acidophilus, Lb. casei, Lb. paracasei and Bifidobacterium spp.

470

and the influence of these bacteria on proteolytic patterns and production of organic acid.

471

International Dairy Journal 2006, 16, 446-456.

472

18. Chun, J.; Kim, K. Y.; Lee, J.-H.; Choi, Y., The analysis of oral microbial communities of

473

wild-type and toll-likereceptor 2-deficient mice using a 454 GS FLX Titanium

474

pyrosequencer. BMC Microbiol 2010, 10, 101.

475

19. Hur, M.; Kim, Y.; Song, H.-R.; Kim, J. M.; Choi, Y. I.; Yi, H., Effect of genetically

476

modified Poplars on soilmicrobial communities during the phytoremediation of waste

477

mine tailings. Appl. Environ. Microbiol. 2011, 77, 7611-7619.

478

20. Kim, B.-S.; Kim, J. N.; Yoon, S.-H.; Chun, J.; Cerniglia, C. E., Impact of enrofloxacin

479

on the human intestinalmicrobiota revealed by comparative molecular analysis.

480

Anaerobe. 2012, 18 (3), 310-320.

481 482

21. Huber, T.; Faulkner, G.; Hugenholtz, P. B., A program to detectchimeric sequences in multiplesequence alignments. Bioinformatics 2004, 20, 2317-2319.

483

22. Kim, O.-S.; Cho, Y.-J.; Lee, K.; Yoon, S.-H.; Kim, M.; Na, H.; Park, S.-C.; Jeon, Y. S.;

484

Lee, J.-H.; Yi, H.; Won, S.; Chun, J., Introducing Eztaxon-e: a prokaryotic 16S rRNA

485

gene sequence database with phylotypes that represent uncultured species. Int. J. Syst.

486

Evol. Microbiol. 2012, 62, 716-721.

487 488

23. Ardö, Y.; Polychroniadou, A., Laboratory manual for chemical analysis of cheese. European Communities: Belgium, 1999.

489

24. Oh, N. S.; Lee, J. Y.; Oh, S.; Joung, J. Y.; Kim, S. G.; Shin, Y. K.; Lee, G. W.; Kim, S. H.;

490

Kim, Y., Improved functionality of fermented milk is mediated by the synbiotic

491

interaction between Cudrania tricuspidata leaf extract and Lactobacillus gasseri strains.

492

Applied Microbiology and Biotechnology 2016, DOI 10.1007/s00253-016-7414-y.

493

25. Jung, J. Y.; Lee, S. H.; Lee, H. J.; Jeon, C. O., Microbial succession and metabolite

494

changes during fermentation of saeu-jeot: Traditional Korean salted seafood. Food

495

Microbiol. 2013, 34 (2), 360-368. 24


Page 24 of 38

Page 25 of 38

496 497


26. McSweeney, P. L. H.; Fox, P. F., Metabolism of residual lactose and of lactate and citrate. 3 ed.; Elsevier Academic Press: London, UK, 2004; Vol. 1.

498

27. Lynch, C. M.; Muir, D. D.; Banks, J. M.; McSweeney, P. L. H.; Fox, P. F., Influence of

499

adjunct cultures of Lactobacillus paracasei ssp. paracasei or Lactobacillus plantarum

500

on Cheddar cheese ripening. Journal of Dairy Science 1999, 82, 1618-1628.

501 502

28. Fox, P. F.; McSweeney, P. L. H.; Cogan, T. M.; Guinee, T. P., Fundamentals of Cheese Science. 1 ed.; Springer US: Gaithersburg, Maryland, USA, 2000.

503

29. Elferink, S. J. W. H. O.; Krooneman, J.; Gottschal, J. C.; Spoelstra, S. F.; Faber, F.;

504

Driehuis, F., Anaerobic conversion of lactic acid to acetic acid and 1,2-propanediol by

505

Lactobacillus buchneri. Applied and Environmental Microbiology 2001, 67 (1), 125-132.

506

30. McGregor, J. U.; White, C. H., Effect of enzyme treatment and ultrafiltration on the

507

quality of low fat Cheddar cheese. Journal of Dairy Science 1990, 73 (3), 571-578.

508

31. Bouzas, J.; Kantt, C. A.; Bodyfelt, F.; Torres, J. A., Simultaneous determination of sugars

509

and organic acids in Cheddar cheese by high-performance liquid chromatography.

510

Journal of Food Science 1991, 56 (1), 276–278.

511 512 513 514 515 516

32. Hugenholtz, J., Citrate metabolism in lactic acid bacteria. FEMS Microbiology Reviews 1993, 12 (1-3), 165-178. 33. Akalin, A. S.; Gönç, S.; Akbaş, Y., Variation in organic acids content during ripening of pickled white cheese. Journal of Dairy Science 2002, 85 (7), 1670-1676. 34. Aston, J. W.; Dulley, J. R., Cheddar cheese flavour. Australian Journal of Dairy Technology 1982, 47, 59-64.

517

35. Hutkins, R. W., Metabolism of starter cultures. CRC Press: New York, NY, 2001.

518

36. Ocando, A. F.; Granados, A.; Basanta, Y.; Gutierrez, B.; Cabrera, L., Organic acids of

519

low molecular weight produced by lactobacilli and enterococci isolated from Palmita-

520

type Venezuelan cheese. Food Microbiology 1993, 10 (1), 1-7.

521

37. Thomas, T. D., Role of lactic acid bacteria and their improvement for production of 25



522

better fermented animal products. New Zealand journal of dairy science and technology

523

1985, 20, 1-10.

524 525

38. Califano, A. N.; Bevilacqua, A. E., Freezing low moisture Mozzarella cheese: changes in organic acid content. Food chemistry. 1999, 64 (2), 193-198.

526

39. Crow, V.; Curry, B.; Hayes, M., The ecology of non-starter lactic acid bacteria (NSLAB)

527

and their use as adjuncts in New Zealand Cheddar. International Dairy Journal 2001, 11

528

(4-7), 275-283.

529 530 531 532 533 534

40. Jordan, K. N.; Cogan, T. M., Identification and growth of non-starter lactic acid bacteria in Irish Cheddar cheese. Irish Journal of Agricultural Food Research 1993, 32, 47-55. 41. Sherwood, I. R., The bacterial flora of New Zealand Cheddar cheese. Journal of Dairy Research 1939, 3, 426-448. 42. Lane, C. N.; Fox, P. F., Contribution of starter and adjunct lactobacilli to proteolysis in Cheddar cheese during ripening. International Dairy Journal 1996, 6 (7), 715-728.

535

43. Pedersen, T. B.; Ristagno, D.; McSweeney, P. L. H.; Vogensen, F. K.; Ardö, Y., Potential

536

impact on cheese flavour of heterofermentative bacteria from starter cultures.

537

International Dairy Journal 2013, 33, 112-119.

538

44. Sadat-Mekmene, L.; Genay, M.; Atlan, D.; Lortal, S.; Gagnairea, V., Original features of

539

cell-envelope proteinases of Lactobacillus helveticus. A review. International Journal of

540

Food Microbiology 2011, 146 (1), 1-13.

541

45. Gagnaire, V.; Carpino, S.; Pediliggieri, C.; Jardin, J.; Lortal, S.; Licitra, G.,

542

Uncommonly thorough hydrolysis of peptides during ripening of Ragusano cheese

543

revealed by tandem mass spectrometry. Journal of Agricultural and Food Chemistry

544

2011, 59 (23), 12443-12452.

545

46. Losito, I.; Carbonara, T.; De Bari, M. D.; Gobbetti, M.; Palmisano, F.; Rizzello, C. G.;

546

Zambonin, P. G., Identification of peptides in antimicrobial fractions of cheese extracts

547

by electrospray ionization ion trap mass spectrometry coupled to a two-dimensional

548

liquid chromatographic separation. Rapid Communications in Mass Spectrometry 2006, 26


Page 26 of 38

Page 27 of 38

549


20, 447-455.

550

47. Lozo, J.; Strahinic, I.; Dalgalarrondo, M.; Chobert, J. M.; Haertlé, T.; Topisirovic, L.,

551

Comparative analysis of β-casein proteolysis by PrtP proteinase from Lactobacillus

552

paracasei subsp. paracasei BGHN14, PrtR proteinase from Lactobacillus rhamnosus

553

BGT10 and PrtH proteinase from Lactobacillus helveticus BGRA43. International

554

Dairy Journal 2011, 21, 863-868.

555

48. Miguel, M.; Recio, I.; Ramos, M.; Delgado, M. A.; Aleixandre, M. A., Antihypertensive

556

effect of peptides obtained from Enterococcus faecalis-fermented milk in rats. Journal of

557

Dairy Science 2006, 89, 3352-3359.

558

49. Møller, K. K.; Rattray, F. P.; Ardö, Y., Application of selected lactic acid bacteria and

559

coagulant for improving the quality of low-salt Cheddar cheese: Chemical,

560

microbiological and rheological evaluation. International Dairy Journal 2013, 33, 163-

561

174.

562

50. Torres-Llanez, M. J.; González-Córdova, A. F.; Hernandez-Mendoza, A.; Garcia, H. S.;

563

Vallejo-Cordoba, B., Angiotensin-converting enzyme inhibitory activity in Mexican

564

Fresco cheese. Journal of Dairy Science 2011, 94, 3794-3800.

565 566

27



Page 28 of 38

Table 1. Chemical compositions (%) of Gouda cheese. Protein GCC1

GCP1

GCP2

Fat

Lactose

Moisture

S/M2

FDM

MNFS

0 wk

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±0.33a

8 wk

25.50±0.12a

30.65±0.69a

N.D.

40.06±0.25b

4.37±0.17a

51.13±1.16a

58.27±0.35b

0 wk

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±1.14a

8 wk

25.34±0.16ab

30.54±0.33a

N.D.

40.24±0.39b

4.33±0.11a

51.96±1.76a

57.92±0.56b

0 wk

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±0.72a

8 wk

25.52±0.16a

30.57±0.52a

N.D

40.71±0.08b

4.33±0.00a

51.56±0.88a

58.63±0.12b

1

GCC = Gouda cheese control, GCP1 = Gouda cheese made with a probiotics1 (Lb. plantarum H4) adjunct culture, GCP2 = Gouda cheese made with a probiotics2 (Lb. fermentum H9) adjunct culture. 2 S/M = Salt in moisture, FDM = Fat in dry matter, MNFS = Moisture in non-fat substance. Values are presented as mean ± SD (n = 3). Data followed by a different lower-case letter within columns were significantly different (P < 0.05).

28


Page 29 of 38


Table 2. Organic acid level of Gouda cheese made with a probiotics adjunct culture during ripening period.

GCC1

GCP1

GCP2

0 wk

Lactic acid (g/kg of cheese) 26.6 ± 0.3ab

Citric acid (mg/kg of cheese) 452.5 ± 3.3h

Acetic acid (mg/kg of cheese) 795.7 ± 13.7a

2 wk

26.1 ± 0.1c

763.1 ± 26.5d

660.3 ± 1.6g

53.9 ± 0.1i

N.D.

4 wk

24.2 ± 0.1e

916.2 ± 32.9b

675.9 ± 6.1f

106.9 ± 0.8g

1.4 ± 0.1e

N.D.

6 wk

d

25.6 ± 0.1

1066.6 ± 0.5

c

751.2 ± 5.2

e

161.0 ± 0.6

d

18.4 ± 3.4

N.D.

8 wk

24.4 ± 0.1e

1044.9 ± 19.3a

712.1 ± 0.1e

179.2 ± 0.5de

N.D.

N.D.

0 wk

a

26.7 ± 0.4

f

594.5 ± 14.6

720.0 ± 4.8

2 wk

26.7 ± 0.1a

762.1 ± 26.8d

4 wk

26.2 ± 0.1bc

6 wk

a

Pyruvic acid (mg/kg of cheese) 41.8 ± 0.3i

de

h

Propionic acid (mg/kg of cheese) N.D.

Formic acid (mg/kg of cheese) 7.8 ± 0.1h N.D.

78.2 ± 4.0

d

15.2 ± 1.0

11.0 ± 0.1g

785.3 ± 1.8a

87.5 ± 2.1h

12.4 ± 1.0e

22.0 ± 0.4d

905.3 ± 13.8b

767.4 ± 1.2b

129.7 ± 0.8g

19.1 ± 1.2d

38.3 ± 0.2c

21.7 ± 0.2d

749.1 ± 19.5d

646.6 ± 7.3g

220.5 ± 3.3b

118.5 ± 9.1c

51.9 ± 1.7a

8 wk

22.8 ± 0.2e

852.4 ± 18.3c

710.3 ± 0.1e

195.9 ± 1.8c

129.1 ± 8.0b

44.7 ± 1.8b

0 wk

24.5 ± 0.2e

406.4 ± 2.6i

733.4 ± 5.7d

85.7 ± 1.2h

N.D.

21.9 ± 0.3d

2 wk

25.3 ± 0.3c

639.0 ± 0.6e

689.2 ± 2.5f

102.7 ± 4.5g

16.3 ± 2.7d

19.7 ± 2.1e

4 wk

21.0 ± 0.1h

469.3 ± 19.3h

614.7 ± 0.6h

172.3 ± 11.6d

129.8 ± 4.4b

20.6 ± 1.2de

6 wk

19.9 ± 0.3i

477.4 ± 12.1h

485.7 ± 7.0i

196.5 ± 16.6c

134.4 ± 6.1b

13.8 ± 0.1f

8 wk 19.2 ± 0.1j 529.7 ± 26.2g 489.4 ± 16.7i 233.4 ± 8.2a 177.8 ± 2.6a 9.9 ± 1.4g 1 GCC = Gouda cheese control, GCP1 = Gouda cheese made with a probiotics1 (Lb. plantarum H4) adjunct culture, GCP2 = Gouda cheese made with a probiotics2 (Lb. fermentum H9) adjunct culture. Values are presented as mean ± SD (n = 3). Data followed by a different lower-case letter within columns were significantly different (P < 0.05). 29



Page 30 of 38

Table 3. Number of sequences, observed diversity richness (OTUs), and diversity estimates of bacteria in Gouda cheese during ripening period. No. of Seq. GCC1

GCP1

GCP2

OTUs

Chao 1

Ace

Shannon

Simpson

0 wk

7622

60

62

61.87

2.96

0.10

2 wk

8466

48

49

49.49

2.85

0.09

4 wk

8542

59

61

60.59

2.94

0.08

6 wk

7773

69

80

81.25

2.76

0.12

8 wk

7828

56

63

62.01

2.97

0.09

0 wk

7961

80

84

84.65

2.89

0.10

2 wk

7236

68

70

71.22

3.08

0.07

4 wk

6985

85

88

88.51

3.35

0.05

6 wk

6627

88

97

96.63

3.32

0.07

8 wk

5854

68

73

76.85

3.17

0.06

0 wk

7547

67

79

72.56

3.07

0.07

2 wk

6709

71

82

77.59

3.09

0.07

4 wk

7085

86

97

93.47

3.33

0.06

6 wk

7262

82

93

92.73

3.13

0.07

8 wk 7182 80 86 86.33 2.96 0.11 GCC = Gouda cheese control, GCP1 = Gouda cheese made with a probiotics1 (Lb. plantarum H4) adjunct culture, GCP2 = Gouda cheese made with a probiotics2 (Lb. fermentum H9) adjunct culture. 1

30


Page 31 of 38


Table 4. Taxonomic classification of the bacterial 16S rRNA gene sequences at genus level (relative abundance>0.01%) of Gouda cheese during ripening period. GCC1 Phylum

Order

Family

GCP1

GCP2

Genus (%) 0wk

2wk

4wk

6wk

8wk

0wk

2wk

4wk

6wk

8wk

0wk

2wk

4wk

6wk

8wk

Firmicutes

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

Firmicutes

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

Firmicutes

Lactobacillales

Streptococcaceae

Unclassified

-

-

-

0.013

-

-

-

-

-

-

-

-

-

-

-

Firmicutes

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

Firmicutes

Lactobacillales

Leuconostocaceae

Unclassified

-

-

0.012

-

-

-

-

-

0.015

-

-

-

-

-

Firmicutes

Lactobacillales

Lactobacillaceae

Lactobacillus

-

-

-

-

-

0.35

0.58

1.32

1.49

1.79

1.40

2.28

1.92

2.16

1.56

Firmicutes

Lactobacillales

Lactobacillaceae

Unclassified

-

-

-

-

-

-

-

0.057

0.045

0.017

-

0.030

0.014

0.041

0.014

Firmicutes

Lactobacillales

Unclassified

Unclassified

-

-

-

-

-

-

-

-

-

-

0.013

-

0.028

-

-

Proteobacteria

Xanthomonadales

Xanthomonadaceae

Stenotrophomonas

-

-

-

-

-

-

-

-

-

-

-

-

-

-

0.014

1

GCC = Gouda cheese control, GCP1 = Gouda cheese made with a probiotics1 (Lb. plantarum H4) adjunct culture, GCP2 = Gouda cheese made with a probiotics2 (Lb. fermentum H9) adjunct culture.

31


0.014


Page 32 of 38

Table 5. Peptide profile of Gouda cheese derived from α-, β-, and κ-casein. GCC1

GCP1

GCP2

GPVRGPFP

●

●

●

96 - 102

ARHPHPH

●

●

●

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 1392

β-Casein β-Casein

193 – 204 194 - 206

YQEPVLGPVRGP

●

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

m/z

Protein

Position

826

β-Casein

199 – 206

851

κ-Casein

862

Sequence

●

● ● ●

●

● ● ●

VSKVKEAMAPKHKEMPFPKYPVEPF

1

●

●

●

GCC = Gouda cheese control, GCP1 = Gouda cheese made with a probiotics1 (Lb. plantarum H4) adjunct culture, GCP2 = Gouda cheese made with a probiotics2 (Lb. fermentum H9) adjunct culture.

32


Page 33 of 38


Figure legend

Figure 1. Taxonomic classification of (A) total bacteria and (B) SLAB bacterial reads at species level retrieved from pooled DNA amplicons from Gouda cheese made with a probiotics adjunct culture during ripening period. Figure 2. Taxonomic classification of (A) NSLAB bacterial reads at species level retrieved from pooled DNA amplicons and (B) the proportion of adjunct probiotics from Gouda cheese made with a probiotics adjunct culture during ripening period. Figure 3. Heat map showing the relative abundances and distribution of representative 16s rRNA gene tag sequences classified at species level. The color code indicates differences in the relative abundance from the mean, ranging from red (negative) through black (the mean) to green (positive). Figure 4. Canonical correspondence analysis (CCA) ordination diagram of bacterial communities associated with (A) chemical compositions and (B) organic acid.

33



Figure 1.

34


Page 34 of 38

Page 35 of 38


Figure 2.

35



Figure 3.

36


Page 36 of 38

Page 37 of 38


Figure 4.

37



TOC Graphic

38


Page 38 of 38

Characterization of the Microbial Diversity and Chemical Composition

Recommend Documents