Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE

Accelerated ripening of Kedong sufu with autochthonous starter cultures Kocuria rosea KDF3 and its protease KP3 as adjuncts Z. Feng, H. Chen, X.T. Lv, H.L. Deng, X. Chen, J.J. Li and L. Guo Key Laboratory of Dairy Science, Ministry of Education, College of Food Science, Northeast Agricultural University, Harbin, Heilongjiang, China

Keywords acceleration, autochthonous adjunct culture, Kedong sufu, Kocuria rosea, protease, proteolysis, ripening. Correspondence Zhen Feng, Key Laboratory of Dairy Science, Ministry of Education, College of Food Science, Northeast Agricultural University, 59 Mucai Road, Harbin 150030, Heilongjiang, China. E-mail: [email protected] 2013/1803: received 4 September 2013, revised 20 December 2013 and accepted 20 December 2013 doi:10.1111/jam.12433

Abstract Aims: Application of autochthonous strain Kocuria rosea KDF3 and its protease KP3 as adjuncts for acceleration of Kedong sufu ripening. Methods and Results: Kedong sufu was manufactured using autochthonous cultures (batch A), K. rosea KDF3 plus autochthonous cultures (batch B) and protease KP3 plus autochthonous cultures (batch C). The effects of certain key factors on the quality of sufu were analysed during a 150-day ripening period. The physicochemical properties of sufu samples from batches B and C after 120 days of ripening met the national standard requirements and samples from batch A after 150 days of ripening. The sensory evaluations of sufu samples from batches B and C after 120 days of ripening and from batch A after 150 days of ripening showed no significant differences (P > 0
05). Furthermore, the mechanism underlying the shorter ripening time and typical sensory quality of sufu prepared with K. rosea KDF3 or protease KP3 was partly revealed by profiles of peptides and free amino acids. The maturation times of Kedong sufu were shortened by 30 days, and the desired characteristics were obtained by adding K. rosea KDF3 or its protease KP3. Conclusions: Kocuria rosea KDF3 or its protease KP3 can hasten sufu maturation. They could be used as adjuncts or additives for accelerating the ripening of Kedong sufu. Significance and Impact of the Study: This study is the first report of using autochthonous strain K. rosea KDF3 or its protease KP3 as adjuncts for accelerating Kedong sufu ripening. The results are useful for characterizing the ripening of Kedong sufu, and they lay the foundation for pilot plant tests and full-scale plant tests.

Introduction Sufu is a traditional fermented soybean product in China. It is a soft, creamy, cheese-like product made from cubes of soybean curd by microbial action. Sufu is similar to cheese in terms of the processing technology and ripening mechanism, and sufu can be used in the same way as cheese in food preparation. Thus, sufu is regarded as the ‘Chinese cheese’ (Steinkraus 1996). It has been widely consumed by Chinese people as an appetizer for more than 1000 years. Sufu can be divided into four groups based on the manufacturing process: mould-fermented

sufu, enzymatically ripened sufu, naturally fermented sufu and bacteria-fermented sufu. Kedong sufu is a typical bacteria-fermented sufu (Wang et al. 2009). The ripening time of Kedong sufu is at least 5 months, so shortening the ripening time without losing any important characteristics would be very advantageous. Because the maturation of sufu adds to the cost of production, any reduction in maturation time would mean significant savings for the sufu industry. Sufu is similar to cheese in terms of processing technology and ripening mechanism. Thus, studies on accelerating cheese ripening could provide useful insights. Different methods

Journal of Applied Microbiology 116, 877--889 © 2014 The Society for Applied Microbiology

877

Z. Feng et al.

Accelerated ripening of Kedong sufu

have been employed to shorten cheese maturation times, including the use of adjunct cultures and the addition of exogenous enzymes (Azarnia et al. 2011). However, nonautochthonous starter cultures can have a negative impact on the sensory properties of the product (Samelis et al. 1998). Hence, autochthonous starter cultures are necessary to achieve the desired fermentation parameters for a specific product type (Erkkil€a et al. 2001). Recent studies have tested the addition of autochthonous strains as starters or adjunct cultures and have analysed the effects on the chemical, microbiological and sensory characteristics of cheese after ripening, and they have demonstrated the beneficial effects of the added cultures (Pisano et al. 2007; Mangia et al. 2008). These results encouraged the use of autochthonous strains in food production to better manage and maintain the ‘typicality’ of fermented food. Similarly, the use of proteases obtained from autochthonous starter cultures isolated from specific fermented food could be more appropriate than the use of exogenous enzymes and commercial enzymes (De Carvalho 2011). Proteolysis provides a range of small-sized peptides and free amino acids, which contribute to the background flavour of most fermented food varieties. Furthermore, free amino acids are important precursors for a range of catabolic reactions, which produce volatile compounds essential for flavour. During the ripening of sufu, soybean proteins break down into small peptides and free amino acids, mainly due to the action of proteases coming from different micro-organisms present in sufu. The peptides and amino acids can in turn be degraded into catabolic products (Han et al. 2004). The high proteolytic activity exerted by the starter cultures would help increase the concentration of small peptides and amino acids, and it would also contribute to texture. In terms of sufu and cheese, the peptides and free amino acids released by proteolysis are important for maturation (Han et al. 2004; Michaelidou et al. 2007; Milesi et al. 2011). Many cheeseisolated strains have been proposed as adjunct cultures based on their contribution to proteolysis and flavour formation in some types of cheese (Milesi et al. 2011). However, little information about the effects of adjunct starter cultures on sufu ripening is currently available. The identification of versatile cultures or enzymes that maintain their desirable properties in the presence of other starter cultures and enzymes would be useful for the food industry and its suppliers. In our previous work, one dominant autochthonous strain, Kocuria rosea KDF3, was isolated from the traditional Kedong sufu (Feng et al. 2013b). Our tests showed that K. rosea KDF3 and its protease KP3 are able to hydrolyse soybean proteins. To our knowledge, the acceleration of sufu ripening by autochthonous starter strains or proteases derived from such 878

strains has not been reported. The objective of this study is to investigate the effects of K. rosea KDS3 and protease KP3 as adjuncts for the acceleration of Kedong sufu ripening. The overall aim is to lay a foundation for the practical use of K. rosea KDS3 or its protease KP3 for the acceleration of sufu ripening. Materials and methods Preparation of starter culture and protease Kocuria rosea KDF3 was isolated from traditional Kedong sufu, as originally reported by Feng et al. (2013b). To prepare starter cultures, K. rosea KDF3 was grown in soybean casein digest broth (Becton, Dickinson and Company, Sparks, MD) at 30°C for 96 h. The cells were subsequently harvested by centrifugation (8000 g for 20 min at 4°C), washed twice with 0
1 mol l 1 sodium phosphate buffer (pH 7), resuspended in the same buffer and carefully adjusted to achieve a concentration of 108 cells ml 1. To prepare autochthonous cultures, sufu samples after 7 days of prefermentation were mixed with 0
1 mol l 1 sodium phosphate buffer (pH 7) (1 : 10, w/w) using a blender. The cells were subsequently harvested by centrifugation (8000 g for 20 min at 4°C), washed twice with 0
1 mol l 1 sodium phosphate buffer (pH 7), resuspended in the same buffer and carefully adjusted to achieve a concentration of 108 cells ml 1. To produce protease KP3, K. rosea KDF3 was cultured in a medium containing 7 g l 1 K2HPO4, 3 g l 1 KH2PO4, 0
1 g l 1 MgSO4, 10 g l 1 yeast extract and 80 g l 1 skim milk powder. This culture was incubated on an orbital shaker (200 rev min 1) for 96 h at 30°C. Purification of the protease KP3 was conducted according to the method of Feng et al. (2013a). The purified protease KP3 was lyophilized. Preparation of Kedong sufu The Kedong sufu was made at a sufu plant in Kedong (Heilongjiang, China). Starting with soybeans, soymilk was obtained after soaking, hot grinding, sieving and cooking. Tofu (soybean curd) was obtained by the coagulation from soymilk. At the same time, protease KP3 was added to soymilk with coagulator (MgCl2). The tofu was pressed to discharge soybean whey and to obtain a firm consistency. The tofu was cut into pieces (3
3 9 3
3 9 1
5 cm3). The salt was spread on the surface of the tofu pieces to increase the salt content to 7%, and it was absorbed over a period of 12 h. The starter culture suspension was sprayed onto the surface of the tofu pieces. The inoculated tofu pieces were placed in plastic trays so that they were evenly spaced. The loaded trays were stacked in an incubation room with

Journal of Applied Microbiology 116, 877--889 © 2014 The Society for Applied Microbiology

Z. Feng et al.

controlled temperature (30°C) and relative humidity (90%). After 7 days, 8 tofu pieces were placed in individual wide-mouth glass bottles with a capacity of 300 ml. Dressing mixture (c. 140 ml) was added to the glass bottles. The dressing mixture consisted of angkak, an alcoholic beverage (rice wine) that produces a final alcohol content of 5%, Chiang (wheat-based miso) and spices. The filled bottles were incubated at 25°C for 150 days. In the fermentation trials, three different batches of sufu were prepared: sufu prepared with autochthonous culture (batch A), with K. rosea KDF3 plus autochthonous culture (1 : 1, v/v) (batch B) and with protease KP3 plus autochthonous culture (batch C). The starter (5
0 9 107 cells ml 1) was used. The protease KP3 (30 mg kg 1) was added. Physicochemical analyses Total acid, moisture, NaCl, amino acid nitrogen and water-soluble protein were measured according to the national standard method (SB/T10170 2007). Briefly, the moisture content of the samples was determined by dehydration at 105°C until the weight remained constant. The total acid content was determined by titration with 0
05 mol l 1 NaOH. The NaCl content was determined by titration with 0
05 mol l 1 AgNO3. The amino acid nitrogen was analysed by the formol and sodium hydroxide titration method. The water-soluble protein content was determined by the trace Kiadhal method. Analysis of peptides The extracts were prepared by mixing sufu samples with water (1 : 1, w/w) in a blender, after which the mixture was centrifuged (7000 g, 20 min). The aqueous phase was recovered. The extracts were deproteinized by mixing them with an equal volume of 24% (w/v) trichloroacetic acid. The mixture was allowed to stand for 10 min and then centrifuged (14 000 g, 10 min). The peptides and free amino acids of the supernatant were measured. Reversed-phase high-performance liquid chromatography (RP-HPLC) was used to separate peptide fractions in an ISCO (ISCO, Lincoln, NE) liquid chromatograph equipped with a UV-visible detector. Samples (20 ll) were loaded into a Spherisorb ODS2 column (C18, 5 lm particle size, 250 9 4
6 mm I.D. ISCO). The process was performed as described previously (Feng et al. 2013a). Peak 1 and Peak 2 of the HPLC results (Fig. 1) from sufu samples obtained after 150 days were analysed by LC-MS to obtain the molecular weight distributions of peptides from Peak 1 and Peak 2. The distributions of the molecular masses of the peptides were determined

Accelerated ripening of Kedong sufu

using an Applied Biosystems MDS Sciex 4800 Plus MALDI TOF (Foster City, CA) according to the method of Cucu et al. (2012). Free amino acid analysis The concentrations of free amino acids were measured on a Beckman 6300 High Performance Amino Acid Analyser (Beckman Instruments Ltd., High Wycombe, UK) fitted with a 120 9 4 mm cation-exchange column (Na+ form). The procedure was performed as described previously (Feng et al. 2013a). Sensory evaluation The sensory properties of sufu were evaluated by 8 panellists from Kedong Sufu Co., Ltd., following standard recommendations (ISO 1993) with a focus on Kedong sufu characteristics. Samples were evaluated from 30 to 150 days of ripening. The flavour, residual intensity flavour, texture, odour, appearance and overall impression of the sufu were evaluated on an arbitrary scale from 1 (bad) to 7 (excellent), with 4 being an acceptable value. These criteria were defined, briefly, as follows: 1) flavour: association of the flavour with the typical characteristics of bacteria-fermented sufu; 2) residual intensity flavour: intensity of flavour at mastication and duration of the flavour; 3) texture: exquisite, soft and uniform texture; 4) odour: intensity of smell and specificity of the smell for this type of product, which should have a slight ester aroma; 5) appearance: external visual impression. Data presentation and statistical analyses Duncan’s multiple range test was used to identify significant differences between batches of sufu. Three replicate tests were performed, and the mean values of measurements were calculated. All results are expressed as the means  standard deviation. Values were considered significantly different when P < 0
05. The data were analysed for the degree of variation, and any significant difference was determined by analysis of variance (ANOVA). All statistical analyses were performed using SAS ver. 8.02 (SAS Institute, Inc., Cary, NC). Results Physicochemical properties Moisture, total acid, NaCl, amino acid nitrogen and water-soluble protein represent important standards for determining the ripeness of sufu (SB/T10170 2007). The

Journal of Applied Microbiology 116, 877--889 © 2014 The Society for Applied Microbiology

879

Z. Feng et al.

Accelerated ripening of Kedong sufu

mAU

mAU

Batch A (30 days)

1000

1000

500

3

4

7

Batch B (30 days) 5

3

500

0

0 0

5 10 15 20 Retention time (min)

5

10 15 20 Retention time (min)

mAU

Batch A (60 days)

mAU

1000

3

6

4

1000

0

25

Batch B (60 days) 5

3

1500

1500

mAU

0

0

0

5 10 15 20 Retention time (min)

25

Batch A (90 days)

1500

3

0

5

10 15 20 Retention time (min)

3

1500

5

Batch B (90 days) 5

2000 mAU

1000

500

500

500

0

0

0

0

0

5 10 15 20 25 Retention time (min) mAU

5

10 15 20 Retention time (min)

mAU

Batch A (120 days)

Batch C (90 days)

1500 4

4

5 10 15 20 25 Retention time (min)

2000 mAU

1000

1000

4

0

25

2

25

4

5 10 15 20 25 Retention time (min) Batch C (120 days)

mAU

Batch B (120 days) 2000

2000

1500

1500

1500

1000

1000

1000

500

500

500

0

0

0

1

0

5

10

1

2

2

15

20

0

25

5

10

15

20

25

0

5

Retention time (min)

Retention time (min) mAU

5

3

0

2000 1

5

3

4 500

25

Batch C (60 days)

1000

500

2000 mAU

5 10 15 20 Retention time (min)

1500

500

0

6 4

0 0

25

Batch C (30 days) 3

4

500

mAU 1000

mAU

Batch A (150 days)

2500

2500

2000

2000

2

2

10

15

20

25

Retention time (min) mAU

Batch B (150 days)

Batch C (150 days)

2500

1

2

2000 1

1

1500

1500

1000

1000

1000

500

500

500

0

0

1500

0

5

10 15 20 Retention time (min)

25

0 0

5

10 15 20 Retention time (min)

25

0

5 10 15 20 Retention time (min)

25

Figure 1 RP-HPLC peptide profiles of the water-soluble fractions of sufu. Batch A, sufu prepared with autochthonous culture; Batch B, sufu prepared with Kocuria rosea KDF3 plus autochthonous culture; Batch C, sufu prepared with protease KP3 plus autochthonous culture.

880

Journal of Applied Microbiology 116, 877--889 © 2014 The Society for Applied Microbiology

Z. Feng et al.

Accelerated ripening of Kedong sufu

total acid, water-soluble protein, amino acid nitrogen, NaCl and moisture values of the three different batches of sufu are shown in Table 1. No significant differences were observed between the batches in terms of moisture and NaCl content. There were only slight differences in the total acid values during ripening. After 150 days of ripening, water-soluble protein was significantly higher in samples from batch B. Samples from batch C were second highest, and samples from batch A had the lowest amount. After 120 days, samples from batch B had similar levels of water-soluble protein compared with batch A at 150 days of ripening. Furthermore, the water-soluble protein content was significantly higher (P < 0
05) in batch C than in batch A at 150 days of ripening. The water-soluble protein content was also significantly higher (P < 0
05) in batch B than in batch C at 120 and 150 days of ripening. After 150 days of ripening, the amino acid nitrogen was significantly higher in samples from batches B and C compared to batch A. After 120 days, samples from batches B and C had similar levels of amino acid nitrogen compared to batch A at 150 days of ripening. Water-soluble protein and amino acid nitrogen were significantly affected by the use of K. rosea KDF3 or its protease KP3. After 120 days of ripening, the levels of moisture, total acid, NaCl, amino acid nitrogen and water-soluble protein in sufu samples from batches B and C met the national standards (SB/T10170 2007). Samples from batch A met the standards after 150 days of ripening.

Peptides RP-HPLC was used to analyse changes in peptides during sufu ripening (Fig. 1). Sufu samples from batches A, B and C showed similar peptide profiles during ripening. From 0 to 90 days, there were 5 major primary peptide peaks (batch A: Peak 3, Peak 4, Peak 5, Peak 6 and Peak 7; batch B: Peak 3, Peak 4 and Peak 5; batch C: Peak 3, Peak 4, Peak 5 and Peak 6) in the RP-HPLC curves of water-soluble extracts that eluted between 9 and 18 min (Fig. 1). At 90 days, only 3 of them remained (Peak 3, Peak 4 and Peak 5). Then, they disappeared at 120 days, while two new peptide peaks formed (Peak 1 and Peak 2), with elution between 9 and 15 min. From 120 to 150 days, the intensities of these two primary peptides increased, without the formation of new peaks. Our results indicate that these two primary peptide peaks (Peak 1 and Peak 2) with retention time value of 9
621 and 13
995 min were characteristic peptide peaks of Kedong sufu prepared with autochthonous starter culture. The results indicated that the effects of K. rosea KDF3 or protease KP3 were relatively small on the RP-HPLC peptide profiles of sufu, whereas autochthonous culture had a greater impact. The differences observed were related to different areasunder-the-curve for the key peptide peaks (Table 2). At 150 days of ripening, total area values of Peak 1 and Peak 2 were highest for the sufu samples from batch B and lowest for the samples from batch A. The total area values of Peak 1 and Peak 2 from batch B at 120 days of maturation were significantly (P < 0
05) higher than those of batch A at

Table 1 Physicochemical changes during fermentation of Kedong sufu and comparison to national standards

Sample Batch A

Batch B

Batch C

SB/T10170-2007

Time (days) 30 60 90 120 150 30 60 90 120 150 30 60 90 120 150

Total acid (g 100 g 1 sufu) 0
44 0
58 0
69 0
84 0
98 0
39 0
52 0
67 0
71 0
82 0
42 0
55 0
67 0
71 0
84 ≤1
2

              

b

0
01 0
02d 0
02ef 0
03g 0
03h 0
01a 0
02c 0
03e 0
02ef 0
03g 0
01ab 0
02cd 0
02e 0
03f 0
03g

Moisture (g 100 g 50  56  58  58  60  49  54  56  57  58  51  56  57  57  59  ≤72

1

sufu)

a

2 2cd 2cd 2cd 3d 2a 2b 2cd 2cd 2cd 1ab 2cd 2cd 2cd 3d

Water-soluble protein (g 100 g 1 sufu)

Amino acid nitrogen (g 100 g 1 sufu)

NaCl (g 100 g

1
21  2
12  2
54  3
03  3
71  1
93  2
72  3
14  3
74  4
73  1
52  2
61  2
93  3
54  4
52  ≥3
5

0
18  0
26  0
29  0
33  0
44  0
23  0
29  0
33  0
45  0
52  0
21  0
27  0
32  0
43  0
51  ≥0
42

7
30 7
35 7
42 7
56 7
61 7
31 7
34 7
44 7
52 7
64 7
32 7
35 7
43 7
57 7
62 ≥6
5

a

0
03 0
05d 0
07e 0
08gh 0
09j 0
06c 0
08f 0
08h 0
11j 0
13l 0
05b 0
07ef 0
07g 0
09i 0
12k

a

0
01 0
01c 0
01d 0
01e 0
02f 0
01b 0
01d 0
01e 0
02f 0
02g 0
01b 0
01cd 0
01e 0
02f 0
02g

              

1

sufu)

0
26a 0
25a 0
28a 0
31a 0
29a 0
23a 0
24a 0
27a 0
32a 0
32a 0
25a 0
22a 0
23a 0
31a 0
33a

Values represent the means  standard deviation. Means within the same columns with different superscripts are significantly different (P < 0
05).

Journal of Applied Microbiology 116, 877--889 © 2014 The Society for Applied Microbiology

881

Z. Feng et al.

Accelerated ripening of Kedong sufu

150 days of ripening. The total area values of Peak 1 and Peak 2 were not significantly different (P > 0
05) between batch A at 150 days of ripening and batch C at 120 days of ripening. However, the total areas of Peak 1 and Peak 2 from sufu samples of batch C were significantly (P < 0
05) higher than in samples from batch A at 150 days of ripening. The total area values of Peak 1 and Peak 2 were also significantly higher (P < 0
05) in batch B than in batch C at 120 days of ripening. The results indicate that the total area values of the two characteristic peptide peaks (Peak 1 and Peak 2) were significantly affected by both K. rosea KDF3 and protease KP3. Mass spectrometry is an effective method for monitoring small peptides produced in sufu as a result of proteolysis. Figure 2 shows the mass spectra of the two characteristic peptide peaks (Peak 1 and Peak 2) with retention time values of 9
621 min and 13
995 min, respectively. Peak 1 and Peak 2 in the HPLC curves from sufu samples at 150 days of ripening were analysed by LC-MS to obtain the molecular weight distributions of peptides. Our aim was to observe proteolysis in sufu from the point of view of the peptides. In addition, low-molecular-weight peptides may produce a savoury or umami taste in a fermented soybean product. For Peak 1, the molecular weight distribution of the dominant peptides ranged from 700 to 877 Da. Furthermore, peptides with molecular masses of 705, 761, 824, 840, 876, 877 Da were dominant. For Peak 2, the molecular weight distribution of the dominant peptides ranges from 705 to 876 Da. Furthermore, peptides with molecular masses of 731, 761, 762, 805, 824, 876 Da were dominant. The production of these low-molecular-weight peptides may be a marker of mature sufu. Free amino acids The concentrations of almost all free amino acids showed a clear tendency to increase with ripening time (Tables 3

and 4). For sufu samples from batch A, the major free amino acids were Pro, Glu, Leu, Phe, Ala, Ile, Tyr and Val. These free amino acids were characteristic of Kedong sufu prepared with autochthonous culture. The major free amino acids were Glu, Leu, Asp, Phe, Pro, Ile, Lys and Val in the sufu samples from batch B. The major free amino acids were Pro, Glu, Leu, Phe, Ala, Lys, Asp and Val in the sufu samples from batch C. Similar distribution patterns of free amino acids were observed for sufu samples from batch B and batch C at the final ripening stages. Compared with sufu samples from batch A, the levels of individual amino acids (Asp, Thr, Ser, Glu, Ala, Val, Met, Ile, Leu, Tyr, Phe, Lys, His and Arg) in sufu were significantly increased after 150 days (P < 0
05) by adding K. rosea KDF3, and the levels of certain amino acids (Asp, Glu, Ala, Val, Ile, Leu, Tyr, Phe, Lys and Arg) were also significantly increased (P < 0
05) by adding protease KP3 (Tables 3 and 4). The levels of individual amino acids (Asp, Thr, Ser, Glu, Val, Met, Ile, Leu, Tyr, Phe, Lys and His) in sufu samples from batch B were significantly higher (P < 0
05) than those of batch C after 150 days of ripening. Furthermore, after fermentation, the levels of essential amino acids were also greatly increased. Sufu samples from batch B contained essential amino acids (Thr, Val, Met, Leu, Phe and Lys) at significantly higher levels than sufu samples from batch A. Sufu samples from batch C contained essential amino acids (Val, Leu, Phe and Lys) at significantly higher levels than sufu samples from batch A, whereas essential amino acids (Thr and Met) had no significant (P > 0
05) differences between batch A and batch C. In this study, sufu produced by K. rosea KDF3 plus autochthonous culture or protease KP3 plus autochthonous culture is a good source of essential amino acids. Total free amino acids significantly increased during ripening in three different batches of sufu samples, with

Table 2 Chromatography peak areas (mAU*s) of key peaks from the three batches of sufu Sample

Time (days)

Batch A

90 120 150 90 120 150 90 120 150

Batch B

Batch C

Peak 1

Peak 2

21 862  1087a 28 382  1363b

17 673  783a 25 054  1192c

c

34 393  1518 37 070  1769d

28 289  1353 33 409  1528e

28 286  1216 33 686  1582c

21 983  987 31 199  1463e

b

Peak 3

Peak 4

Peak 5

Total area of key peaks

17 854  856c

10 660  495a

16 987  819a

12 917  558a

28 213  1332c

16 650  811a

15 140  743b

13 678  657b

15 586  768a

45 39 53 57 62 70 44 50 64

d

b

501 535 436 780 682 479 404 269 885

        

1967b 1752a 2432cd 2658d 2891e 3386f 2046b 2313c 3079e

Total area of key peaks at 90 days of ripening is the sum of Peak 3, Peak 4 and Peak 5. Total area of key peaks at 120 or 150 days of ripening is the sum of Peak 1 and Peak 2. Values represent the means  standard deviation (n = 3). Means within the same columns with different superscripts are significantly different (P < 0
05).

882

Journal of Applied Microbiology 116, 877--889 © 2014 The Society for Applied Microbiology

Z. Feng et al.

Accelerated ripening of Kedong sufu

824·2306

(a) 100

90

90

80

80

70

70

60

761·7884

60

761·6428 % intensity

% intensity

(b) 100

50 876·0526

50

762·7820

824·4135

40

40

731·8127 30

705·6057

877·0699

30

700·6628

705·7438 706·7676 20 708·7770 718·7919 747·7295 10

840·1672 20

723·5579 720·6215

10

0 699

805·7156 876·2956 860·3668 840·4041

0 699

961 Mass (m/z)

961 Mass (m/z)

Figure 2 MS spectra of two characteristic peptide peaks. (a) MS spectrum of Peak 1; (b) MS spectrum of Peak 2.

sufu samples from batch B having the highest amounts of total free amino acids and sufu samples from batch C the second highest after 150 days of maturation (Table 4). The level of total free amino acids in sufu samples from batch B at 120 days of maturation was significantly higher (P < 0
05) than in sufu samples from batch A after 150 days of ripening. No significant (P > 0
05) differences were found between batch A at 150 days of ripening and batch C at 120 days of ripening. However, total free amino acids in sufu samples from batch C were significantly (P < 0
05) higher than in batch A at 150 days of ripening. The total free amino acid levels were also significantly higher (P < 0
05) in batch B than in batch C at 120 and 150 days of ripening. The levels of total free amino acid were significantly affected by the use of autochthonous cultures with K. rosea KDF3 or its protease KP3, and they were significantly affected by ripening time. Sensory evaluation Table 5 shows the sensory characteristics (flavour, residual intensity flavour, texture, odour, appearance and

overall impression) of the three different batches of sufu samples. In general, fully mature Kedong sufu should have the typical characteristics of bacteria-fermented sufu: enduring residual intensity flavour, exquisite, soft and uniform texture, a slightly ester aroma, an integrated appearance and easy dissolution in the mouth. In the three different batches of sufu samples, scores for flavour, residual intensity flavour, texture and odour were below 4 points at 30 and 60 days of ripening (data not shown). Batch A, which was made with autochthonous culture, was used as the control. The best flavour and maximum residual intensity of flavour were first obtained in batch B and batch C at 120 days of ripening, respectively, and in batch A at 150 days. Furthermore, flavour and residual intensity of flavour scores for sufu samples from batches B and C at 120 days of ripening were not significantly different from those of batch A sufu samples at 150 days of ripening. However, the defects of sufu samples from batches B and C included a residual weak bitter taste at 150 days of ripening, which was not observed in batch A. Texture scores were highest for batches B and C at 120 days of ripening, and they were not significantly different to scores obtained at 150 days of ripening. The

Journal of Applied Microbiology 116, 877--889 © 2014 The Society for Applied Microbiology

883

884

30 60 90 120 150 30 60 90 120 150 30 60 90 120 150

Batch A

48
94 65
82 83
77 116
0 98
3 112
3 175
6 182
9 244
9 305
7 103
9 114
3 162
6 130
3 164
8

Asp

               2
37 2
57b 3
35c 4
70e 3
33d 4
63e 7
71hi 8
19i 10
46j 13
2k 4
73de 4
98e 6
93g 5
88f 6
87gh

a

43
92 38
26 58
61 64
65 67
28 83
72 97
37 129
61 140
9 135
5 55
61 52
72 62
88 63
27 67
56

Thr*                31
91 20
62 44
13 31
31 38
42 70
11 142
4 74
83 131
6 99
58 56
61 32
12 48
59 43
91 31
51

Ser                1
26 1
13a 2
03d 1
36b 1
55c 3
33f 5
75j 3
14g 5
32i 4
39h 2
17e 1
13b 1
98d 1
87d 1
33b

b

325
4 323
1 388
2 422
6 373
6 553
9 729
1 699
9 881
7 1001
3 482
3 484
5 556
3 507
1 515
8

Glu                13
6 13
2a 16
3bc 17
6c 13
2b 25
6e 29
7f 27
6f 38
3g 41
2h 19
2d 18
9d 23
7e 21
3d 21
9de

a

59
19 90
01 94
49 122
4 116
7 75
93 75
92 89
72 123
3 135
4 91
41 116
9 118
4 131
1 132
9

Gly

sufu) during fermentation of Kedong sufu

2
52 1
36a 2
87bc 2
98cd 3
16d 3
73e 4
16f 5
28g 6
35h 5
77gh 2
12b 2
03b 2
66cd 2
74cd 2
96d

a

1

Values represent the mean  standard deviation. Means within the same columns with different superscripts are significantly different (P < 0
05). *Essential amino acid.

Batch C

Batch B

Time (days)

Sample

Table 3 Changes in free amino acid content (mg 100 g

               2
97 3
62bc 4
52bcd 5
37def 4
38cde 2
69ab 2
41ab 3
78bc 5
17def 4
87ef 3
82bc 4
67cde 5
12cde 5
77ef 5
78ef

a

95
22 125
5 120
7 162
6 181
5 73
25 135
5 170
5 195
6 207
8 130
2 149
4 182
1 169
5 212
8

Ala                4
27 5
78cd 4
58c 7
32f 7
32gh 2
57a 5
92d 7
62fgh 7
98i 9
77j 5
52cd 6
27e 7
96h 7
73fg 9
34j

b

0
5337 0
1751 43
11 73
66 95
82 7
832 13
03 14
54 10
62 18
41 42
35 51
47 64
77 75
14 87
65

Cys               

0
018 0
008a 1
91e 3
22h 3
82j 0
221b 0
521c 6
67cd 0
461bc 0
882d 1
66e 2
38f 3
11g 3
71h 3
39i

a

126
9 108
8 112
3 150
1 140
1 118
7 144
2 156
0 187
6 208
6 120
5 141
1 153
9 164
9 162
7

Val*               

5
90 4
10a 4
31a 7
12cde 5
97c 4
21ab 5
33cd 6
94ef 8
63g 8
93h 5
63ab 5
94c 6
68def 7
79f 7
35f

b

21
26 24
31 28
31 31
52 31
68 30
61 38
76 39
81 51
63 55
15 28
52 30
98 30
47 39
22 32
4

Met*               

1
05a 1
12b 1
37c 1
52d 1
37d 1
22cd 1
67e 1
32e 2
11f 2
36g 1
16c 1
24cd 1
18cd 1
43e 1
22d

Accelerated ripening of Kedong sufu Z. Feng et al.

Journal of Applied Microbiology 116, 877--889 © 2014 The Society for Applied Microbiology

Z. Feng et al.

Discussion

1388 1860 2021 2633 2735 1986 2617 2672 3467 3749 1952 2434 2642 2798 3143 200
3 475
9 366
6 639
7 753
9 15
66 128
1 22
52 185
6 232
1 247
2 491
2 481
2 562
7 570
3 0
127 0
183bc 0
135ab 0
225bc 0
012a 0
461d 8
51e 0
321d 0
321cd 0
186bc 0
134ab 0
121ab 0
173bc 0
083ab 0
157ab 0
9992 1
231 3
761 1
942 5
791 23
22 26
63 30
08 36
64 36
02 1
542 1
531 1
622 2
453 1
481                               98
03 121
1 145
2 168
7 169
7 172
9 197
8 238
5 269
3 289
6 127
1 165
1 156
8 195
0 274
9 3
21 4
81b 5
82c 6
63ef 6
35de 4
91d 6
72fg 6
77fgh 8
64i 7
12h 4
86b 6
32de 6
18d 6
94gh 6
55fg

               82
33 104
7 120
2 157
4 148
9 143
6 160
9 168
4 197
8 177
4 108
1 146
5 143
6 169
9 160
6 5
21 8
64b 8
64c 9
72ef 7
32ef 10
1d 9
71fg 12
2h 15
4i 18
5j 7
03b 8
68de 9
13de 10
3g 15
2i

               127
0 183
7 211
1 249
7 254
3 229
8 265
3 304
8 372
4 399
6 178
2 236
1 240
8 277
7 362
6 2
72 3
92bc 4
31c 6
65d 5
85d 5
52d 4
91d 6
73e 9
68f 9
73g 4
52b 5
56d 6
79d 7
12e 6
97e

               70
98 108
6 117
5 145
7 144
8 136
2 136
4 169
6 211
6 225
1 104
3 134
7 144
3 159
1 158
8 Batch C

Batch B

30 60 90 120 150 30 60 90 120 150 30 60 90 120 150 Batch A

Values represent the mean  standard deviation. Means within the same columns with different superscripts are significantly different (P < 0
05). *Essential amino acid.

52
48 63
00 79
62 89
84 113
9 127
7 129
9 168
9 217
2 216
6 71
14 81
77 88
09 103
9 202
1 3
56 3
85b 6
77c 7
53de 7
68de 6
93e 9
23f 9
25g 10
3h 12
9i 4
98b 6
73de 6
25cd 8
66f 11
6h

Lys*

a

Phe*

a

Tyr

a

Leu*

a

Ile Time (days) Sample

Table 4 Changes in free amino acid concentrations (mg 100 g

1

sufu) during fermentation of Kedong sufu

a

2
13 2
92b 3
58cd 3
83d 4
45f 4
92h 5
77h 7
63i 9
38k 9
52k 2
57bc 3
16d 3
51d 4
69e 8
62j

His

              

a

Arg

              

ab

Pro

              

c

9
72 22
5f 13
5e 28
4h 25
6i 0
661a 4
99b 0
871a 8
37c 8
73d 9
61d 19
4f 20
3f 25
6g 25
9g

Total

               3
462 5
472 3
422 4
913 0
3087 11
22 19
99 11
17 8
722 4
771 3
481 3
422 5
541 2
742 4
363

odour scores were highest in batches B and C at 150 days of ripening. Odour scores of sufu samples from batches B and C at 120 days of ripening had similar scores to batch A at 150 days of ripening. As far as appearance is concerned, scores decreased gradually because of proteolysis. The appearances of sufu samples from batches B and C at 120 days of ripening were better than at 150 days, although the differences were not significant. Notably, the appearances of sufu samples from batches B and C were not integrated at 150 days of ripening. In terms of overall impression, batches B and C were, at 120 days of ripening, rated the best, but at 150 days, batch A had the highest scores. The addition of K. rosea KDF3 or its protease KP3 appears to have improved the sensorial qualities, but the effect although is not very substantial.

0
023 0
042a 0
170ab 0
075a 0
214b 1
13c 5
16d 1
27e 1
57e 1
65e 0
056a 0
049a 0
055a 0
086a 0
063a

57a 86b 93b 101cd 122d 81b 107cd 112d 146f 158g 83b 91c 117d 119d 132e

Accelerated ripening of Kedong sufu

Spontaneous fermentation and natural starter cultures produced largely through backslopping are widely used in traditional fermented food. The latter practice plays a decisive role in determining the typicity of traditional fermented food. In some instances, backslopping is used to initiate and control sufu fermentation. Using an autochthonous strain as an adjunct for the acceleration of fermented food ripening can effectively preserve the typical characteristics of the food. In this study, K. rosea KDF3 and its protease KP3 were used in an attempt to accelerate sufu ripening. The results show that K. rosea KDF3 and its protease KP3 can speed up sufu maturation while maintaining the typical characteristics of Kedong sufu. The proteolytic activity of K. rosea KDF3 and its protease KP3 on soybean protein contributed to these results. The levels of peptides, total free amino acids and 14 of the 17 individual free amino acids were significantly higher (P < 0
05) in sufu samples from batch B at 120 days of ripening than in those of batch A at 150 days of ripening. The levels of amino acid nitrogen and watersoluble protein in samples from batch B were significantly higher (P < 0
05) than in those of batch A at 150 days of ripening. These results indicate that adding K. rosea KDF3 significantly increases the amounts of water-soluble proteins, amino acid nitrogen, characteristic peptides, free amino acids and total free amino acids. Thus, K. rosea KDF3 can be used as an adjunct to speed up sufu maturation. Among strains from the autochthonous microflora of Kedong sufu, K. rosea KDF3 has a strong ability to hydrolyse soybean proteins (Z. Feng, X. Chen, J.J. Li and D. Ren, unpublished data), resulting in higher levels of protein degradation products. Adjunct cultures are widely used to accelerate the maturation of traditional fermented food. Michaelidou et al. (2003a) investigated the effects of adjunct cultures (Lactococcus lactis) on proteolysis in

Journal of Applied Microbiology 116, 877--889 © 2014 The Society for Applied Microbiology

885

Z. Feng et al.

Accelerated ripening of Kedong sufu

Table 5 Sensory characteristics of sufu observed during ripening Sample

Time (days)

Flavour

Batch A

90 120 150 90 120 150 90 120 150

4
2 4
6 4
9 4
4 5
1 4
8 4
3 5
0 4
9

Batch B

Batch C

        

0
1a 0
1bc 0
2de 0
1ab 0
2e 0
1cd 0
1a 0
2de 0
2de

Residual intensity flavour 4
1 4
7 5
2 4
2 5
3 5
1 4
2 5
2 5
0

        

0
1a 0
2b 0
2cd 0
1a 0
2d 0
1cd 0
1a 0
2cd 0
1c

Texture 4
3 4
5 5
1 4
6 5
2 5
0 4
5 5
1 4
9

        

0
1a 0
1a 0
2b 0
1a 0
2b 0
2b 0
1a 0
2b 0
2b

Odour 4
5 4
7 4
9 4
6 5
0 5
5 4
5 4
9 5
3

        

Appearance 0
1a 0
1abc 0
2bc 0
1ab 0
2c 0
2d 0
1a 0
2bc 0
2d

5
2 5
2 4
8 5
1 4
9 4
6 5
1 4
9 4
7

        

0
1d 0
2d 0
2ab 0
2cd 0
1bc 0
1a 0
2cd 0
1bc 0
1ab

Overall impression 6
5 6
8 7
1 6
6 7
2 6
9 6
6 7
1 6
8

        

0
2a 0
2abc 0
2cd 0
2ab 0
2d 0
2bcd 0
2ab 0
2cd 0
2abc

Values represent the mean  standard deviation. Means within the same columns with different superscripts are significantly different (P < 0
05).

cheese. The results showed that the levels of total soluble nitrogen and free amino acids were significantly increased by adding Lc. lactis as an adjunct culture. The effect of two adjunct cultures (LBC 80 and CR-213) on proteolysis in cheese was also investigated. The results showed that the levels of total nitrogen and free amino acids were significantly (P < 0
05) higher in experimentally treated cheeses than in control cheese (Michaelidou et al. 2003b). These studies of cheese fermentation are in good agreement with the results of our sufu study. When protease KP3 is used as an adjunct, the levels of water-soluble proteins, amino acid nitrogen, peptides, total free amino acids and 11 of the 17 individual free amino acids are significantly higher (P < 0
05) in batch C than in batch A at 150 days of ripening. Furthermore, using protease KP3 as an adjunct can speed up sufu maturation. Proteinases are widely used to accelerate cheese maturation. Accelase AM317 and Accelase AHC50 from Lactobacillus lactis were clearly shown to accelerate ripening in cheese by enhancing proteolysis (Kilcawley et al. 2012). The use of natural and recombinant enzymes from Lactobacillus rhamnosus S93 in cheese led to greater proteolysis (Azarnia et al. 2010). These studies are in good agreement with the results of our sufu study. When K. rosea KDF3 or protease KP3 was used, the levels of water-soluble proteins, peptides, 12 of 17 free amino acids and total free amino acid were significantly higher (P < 0
05) in batch B than in batch C at 150 days of ripening. The results can be explained by the water solubility of most enzymes, which may cause the enzymes to be lost in the whey (Azarnia et al. 2006). Microencapsulation of enzymes can be a cost-effective way to ensure uniform delivery of enzymes into the sufu matrix without significant losses into the whey. During ripening, encapsulated enzymes are released into the curd upon capsule breakdown (Azarnia et al. 2011). To avoid the loss of protease KP3, we are now investigating the use of encapsulation. In addition, proteolytic activity is affected by 886

numerous endogenous and exogenous ecological factors, for example process parameters such as temperature, pH, NaCl concentration, alcohol concentration, and the presence of enzymes from other micro-organisms. The released peptides were hydrophilic (RT: 7–15 min), as revealed by the RP-HPLC elution pattern (Aguirre et al. 2008). In the present study, two characteristic peptide peaks (Peak 1 and Peak 2) were eluted, with retention times ranging from 9 to 15 min. Hydrophilic peptides are normally correlated with desirable, fermented soy flavours (Smit et al. 2005), whereas hydrophobic peptides are associated with bitterness. HPLC chromatograms from the end of the ripening period (120 or 150 days) showed that peptide peaks (Peak 5, Peak 6 and Peak 7) with retention times ranging from 15 to 20 min disappeared during ripening. The peptides may have been hydrolysed, resulting in them contributing to the two characteristic peptide peaks (Peak 1 and Peak 2). Thus, we inferred that the disappearance of hydrophobic peptide peaks and the appearance of characteristic peptide peaks might be important markers of sufu maturity. Taste-active oligopeptides are recognized as important contributors to taste in various foods. Mee-Ra and Eun-Young investigated the taste characteristics of doenjang (a traditional Korean fermented soybean food) water extract for component compounds that contribute to its taste. The doenjang water extract was fractionated, and the 1000 Da > MW ≥ 500 Da fraction had the highest peptide levels and elicited the strongest umami taste (Mee-Ra and Eun-Young 2011). In this study, the molecular masses of the peptides of the two characteristic peaks (Peak 1 and Peak 2) ranged from 700 to 900 Da. These low-molecular-weight peptides may contribute to the umami taste of sufu. In traditional fermented soybean products, free amino acids contribute directly to the taste perception and act as precursors of flavour (Lioe et al. 2007; Qin and Ding 2007; Dajanta et al. 2011). Pro and Ala are sweet, Glu is umami, Leu and Phe are bitter. Leu might be an important precursor of branched-chain volatile flavour

Journal of Applied Microbiology 116, 877--889 © 2014 The Society for Applied Microbiology

Z. Feng et al.

compounds (Kato et al. 1989; Schoenberger et al. 2002). Sufu samples from batches B and C had higher levels of Glu, Ala, Leu and Phe than batch A at 150 days of ripening. Glu and its salts are the principal cause of the delicious taste of various fermented soybean foods, such as miso, koji and soy sauce (Kato et al. 1989; Schoenberger et al. 2002). Han et al. (2004) demonstrated that Glu, Leu, Ala and Phe are the predominant free amino acids in mould-fermented sufu. Similar patterns of amino acids were observed in kinema (a traditional Indian fermented soybean food) (Sarkar et al. 1997). Sufu samples from batch B contained essential amino acids at significantly higher levels than sufu samples from batch A. Similar results were reported by Sarkar et al. (1997) and Dajanta et al. (2011). In this study, sufu produced using K. rosea KDF3 with an autochthonous starter culture was a good source of essential amino acids. Sufu samples from the three different batches showed similar distribution patterns of free amino acids and peptides at different time points during ripening. This shows K. rosea KDF3 and protease KP3 have a relatively small effect on the distribution patterns of free amino acids and peptides, whereas autochthonous cultures have a greater impact. The quantities associated with two characteristic peptide peaks (Peak 1 and Peak 2) from sufu samples of batch B and batch C were significantly (P < 0
05) higher than those of batch A at 120 and 150 days of ripening, respectively. For the eight dominant free amino acids (Pro, Glu, Leu, Phe, Ala, Ile, Tyr and Val), some concentrations (Glu, Val, Ile, Leu and Phe) were significantly increased compared to batch A (P < 0
05, 150 days of ripening) by adding K. rosea KDF3, and the concentrations of individual amino acids (Glu, Ala, Val, Leu and Phe) were significantly increased by adding protease KP3 (P < 0
05, 150 days of ripening). The results indicated that K. rosea KDF3 or protease KP3 play important role in amounts of peptides and free amino acids. It is important to be able to estimate the optimal ripening time of fermented food (Sihufe et al. 2010). Currently, the physicochemical properties of sufu must conform to national standards, and sensory evaluations are performed by inspectors to ensure product quality. The physicochemical properties (total acid, water-soluble proteins, amino acid nitrogen, NaCl, moisture) of sufu samples from batches B and C at 120 days of ripening met the national standards (SB/T10170 2007) and the physicochemical properties of sufu samples from batch A at 150 days of ripening (Table 1). Sensory quality control is a very important tool for ensuring that a product has the expected sensory characteristics (Hilde et al. 2012; Etaio et al. 2013). The sensory evaluations of sufu samples from batches B and C at 120 days of

Accelerated ripening of Kedong sufu

ripening and from batch A at 150 days of ripening showed no significant differences. The maturation times of Kedong sufu were shortened by 30 days in batches B and C. Samples from batches B and C at 150 days of ripening presented only increased bitterness and decreased formability compared to the 120-day samples. These effects can be attributed to excessive proteolysis. Similar results were reported for Armada cheese by Herreros et al. (2007). In this study, the maturation time of Kedong sufu was shortened by 30 days by adding K. rosea KDF3 or its protease KP3. Using these adjuncts resulted in controlled acceleration of Kedong sufu maturation, and such adjuncts could be used to accelerate full-scale industrial production of sufu. The results of our study are very encouraging and will lay a foundation for pilot plant tests and full-scale plant tests. To the best of our knowledge, this is the first report of accelerating Kedong sufu ripening by the use of autochthonous adjunct strains or proteases. Acknowledgements This research was supported by the young academic backbone support project of Heilongjiang province (1252G009). Conflict of Interest It should be understood that none of the authors have any financial or scientific conflict of interest with regard to the research described in this manuscript. References Aguirre, L., Garro, M.S. and De Gioria, G.S. (2008) Enzymatic hydrolysis of soybean protein using lactic acid bacteria. Food Chem 111, 976–982. Azarnia, S., Robert, N. and Lee, B. (2006) Biotechnological methods to accelerate Cheddar cheese ripening. Crit Rev Biotechnol 26, 121–143. Azarnia, S., Lee, B.H., Yaylayan, V. and Kilcawley, K.N. (2010) Proteolysis development in enzyme-modified Cheddar cheese using natural and recombinant enzymes of Lactobacillus rhamnosus S93. Food Chem 120, 174–178. Azarnia, S., Lee, B., St-Gelais, D., Kilcawley, K. and Noroozi, E. (2011) Effect of free and encapsulated recombinant aminopeptidase on proteolytic indices and sensory characteristics of Cheddar cheese. LWT-Food Sci Technol 44, 570–575. Cucu, T., De Meulenaer, B. and Devreese, B. (2012) MALDI based identification of soybean protein markers: possible analytical targets for allergen detection in processed foods. Peptides 33, 187–196.

Journal of Applied Microbiology 116, 877--889 © 2014 The Society for Applied Microbiology

887

Z. Feng et al.

Accelerated ripening of Kedong sufu

Dajanta, K., Apichartsrangkoon, A. and Frazier, R.A. (2011) Free-amino acid profiles of thua nao, a Thai fermented soybean. Food Chem 125, 342–347. De Carvalho, C.C.C.R. (2011) Enzymatic and whole cell catalysis: finding new strategies for old processes. Biotechnol Adv 29, 75–83. Erkkil€a, S., Suihko, M.L., Eerola, S., Pet€aj€a, E. and MattilaSandholm, T. (2001) Dry sausage fermented by Lactobacillus rhamnosus strains. Int J Food Microbiol 64, 205–210. Etaio, I., Gil, P.F., Ojed, M., Albisu, M., Salmer on, J. and Perez, E.F.J. (2013) Evaluation of sensory quality of calf chops: a new methodological approach. Meat Sci 94, 105–114. Feng, Z., Chen, X., Li, J.J. and Ren, D. (2013a) An alkaline protease from Kocuria kristinae F7: properties and characterization of its hydrolysates from soy protein. Eur Food Res Technol 236, 293–301. Feng, Z., Gao, W., Ren, D., Chen, X. and Li, J.J. (2013b) Evaluation of bacterial flora during the ripening of Kedong sufu, a typical Chinese traditional bacteriafermented soybean product. J Sci Food Agric 93, 1471–1478. Han, B.Z., Frans, M., Rombouts, M.J. and Robert, N. (2004) Amino acid profiles of sufu, a Chinese fermented soybean food. J Food Compos Anal 17, 689–698. Herreros, M.A., Arenas, R., Sandoval, M.H., Castro, J.M., Fresno, J.M. and Tornadijo, M.E. (2007) Effect of addition of native cultures on characteristics of Armada cheese manufactured with pasteurized milk: a preliminary study. Int Dairy J 17, 328–335. Hilde, K., Steffen, S., Roger, K. and Abrahamsen., (2012) Quality scoring – A tool for sensory evaluation of cheese? Food Qual Prefer 26, 221–230. ISO (1993). Sensorial analysis-General guidance for the selection, training and monitoring of assessors. Standard ISO 8586-1. Geneva, Switzerland: International Organization for Standardization. Kato, H., Rhue, M.R. and Nishimura, T. (1989) Role of free amino acids and peptides in food taste. In Flavor chemistry trends and developments ed. Teranishi, R.R., Buttery, G. and Shahidi, F., pp. 158–174. Washington: American Chemical Society. Kilcawley, K.N., Nongonierma, A.B., Hannon, J.A., Doolan, I.A. and Wilkinson, M.G. (2012) Evaluation of commercial enzyme systems to accelerate Cheddar cheese ripening. Int Dairy J 26, 50–57. Lioe, H.N., Wade, K., Aoki, T. and Yasuda, M. (2007) Chemical and sensory characteristics of low molecular weight fractions obtained from three types of Japanese soy sauce (shoyu) – Koikuchi, tamari and shiro shoyu. Food Chem 100, 1669–1677. Mangia, N.P., Murgia, M.A., Garau, G., Sanna, M.G. and Deiana, P. (2008) Influence of selected lab cultures on the evolution of free amino acids, free fatty acids and Fiore

888

Sardo cheese microflora during the ripening. Food Microbiol 25, 366–377. Mee-Ra, R. and Eun-Young, K. (2011) Umami taste characteristics of water extract of Doenjang, a Korean soybean paste: low-molecular acidic peptides may be a possible clue to the taste. Food Chem 127, 1210–1215. Michaelidou, A., Katsiari, M.C., Kondyli, E., Voutsinas, L.P. and Alichanidis, E. (2003a) Effect of a commercial adjunct culture on proteolysis in low-fat Feta-type cheese. Int Dairy J 13, 179–189. Michaelidou, A., Katsiari, M.C., Voutsinas, L.P., Kondyli, E. and Alichanidis, E. (2003b) Effect of commercial adjunct cultures on proteolysis in low-fat Kefalograviera-type cheese. Int Dairy J 13, 743–753. Michaelidou, A., Katsiari, M.C., Voutsinas, L.P., Polychroniadou, A. and Alichanidis, E. (2007) Effect of multiple-species starters on peptide profile and free amino acids in low-fat Kefalograviera-type cheese. Food Chem 104, 800–807. Milesi, M.M., Bergamini, C.V. and Hynes, E. (2011) Production of peptides and free amino acids in a sterile extract describes peptidolysis in hard-cooked cheeses. Food Res Int 44, 765–773. Ministry of Commerce of the People’s Republic of China (2007) Sufu. Standard of sufu. Ministry of Commerce of the People’s Republic of China SB/T10170. Beijing: Ministry of Commerce of the People’s Republic of China. Pisano, M.B., Fadda, M.E., Deplano, M., Corda, A., Casula, M. and Cosentino, S. (2007) Characterization of Fiore Sardo cheese manufactured with the addition of autochthonous cultures. J Dairy Res 74, 255–261. Qin, L. and Ding, X. (2007) Formation of taste and odor compounds during preparation of Douchiba, a Chinese traditional soy-fermented appetizer. J Food Biochem 31, 230–251. Samelis, J., Metaxopoulos, J., Vlassi, M. and Pappa, A. (1998) Stability and safety of traditional Greek salami—a microbiological ecology study. Int J Food Microbiol 44, 69–82. Sarkar, P.K., Jones, L.J., Craven, G.S., Somerset, S.M. and Palmer, C. (1997) Amino acid profiles of kinema, a soybean-fermented food. Food Chem 59, 69–75. Schoenberger, C., Krottenthaler, M. and Back, W. (2002) Sensory and analytical characterization of nonvolatile taste-active compounds in bottom-fermented beers. Master Brewers Association of the Americas 39, 210–217. Sihufe, G.A., Zorrilla, S.E., Perotti, M.C., Wolf, I.V., Zalazar, C.A., Sabbag, N.G., Costa, S.C. and Rubiolo, A.C. (2010) Acceleration of cheese ripening at elevated temperature. An estimation of the optimal ripening time of a traditional Argentinean hard cheese. Food Chem 119, 101–107.

Journal of Applied Microbiology 116, 877--889 © 2014 The Society for Applied Microbiology

Z. Feng et al.

Smit, G., Smit, B.A. and Engels, W.J.M. (2005) Flavour formation by lactic acid bacteria and biochemical flavour profiling of cheese products. FEMS Microbiol Rev 29, 591–610. Steinkraus, K.H. (1996) Chinese sufu. In Handbook of indigenous fermented foods ed. Steinkraus, K.H., pp. 633– 641. New York: Marcel Dekker.

Accelerated ripening of Kedong sufu

Wang, R.Z., Wei, X.Y., Shen, Z.H., Wu, Z.G., Huang, D.P. and Qu, G.Y. (2009) The characteristic sufu in China. In The production of sufu in China ed. Tu, R.L. and Li, G.G., pp. 230–232. Beijing: Light Industry Press.

Journal of Applied Microbiology 116, 877--889 © 2014 The Society for Applied Microbiology

889