OBJECTIVE The aim of this study is to improve the nutritional and sensorial quality and the shelf life of commercial tofu by lactic acid fermentation. INTRODUCTION Tofu, a fundamental part of Asian food culture, is a traditional oriental soybean food composed principally of protein and oil (Kitawaki et al., 2009). Contemporary, its consume and popularity is growing rapidly in popularity in the West. With the intent to preserve its quality for a longer period of time, tofu is often pasteurized or packed under vacuum. It is possible that these treatments are not enough to prevent the development of spoilage or pathogen microorganisms. Tofu pH is around 6.5 and Aw around 1: these characteristics make it susceptible to spoilage microorganisms affecting shelf-life. Fermentation of soy-milk by probiotics leads to a development of a new more stable functional food with bioactive components. In this study a new way to produce tofu with soy-milk fermentation by probiotic lactic acid bacteria (Lactobacillus casei and Lactobacillus acidophilus) has been developed, in order to prevent undesired microbial growth, improve stability and nutritional quality of the product. Moreover, to confirm the stability of the tofu obtained with fermented soymilk, a challenge test, with microorganisms isolated from contaminated tofu, has been set up. Diana I. Serrazanetti* 1,2 , Maurice Ndagijimana 3 , Claudio Miserocchi 4 , Eleonora Tosato 4 , M. Elisabetta Guerzoni 1,2 1 Department of Food Science, University of Bologna, Via Fanin, 44 – 40127 Bologna, Italy. 2 Inter-departmental Center of Industrial Agri-Food Research (CIRI Agroalimentare), P.zza Goidanich 60, 47521, Cesena, Italy. 3 Department of Agricultural Food & Nutritional Science 410, Agriculture/Forestry Centre University of Alberta, Edmonton, AB, Canada. 4 Con.Bio. s.r.l. Via Del Salice, 42 - 47822 Santarcangelo di Romagna (RN), Italy *[email protected] CONCLUSIONS • The lactic acid fermentation lead to a new tofu (or new ingredient) whit an increased shelf life. • The product obtained can be simply distinguished by the traditional product from the sensorial and analytical point of view. • The lactic acid fermentation can be also interesting for the improved nutritional effects on soy products (conversion of isoflavones in aglicones and antioxidant compounds production) (Ruy et al., 2010). Innovative methods of tofu production by soy milk fermentation. Enhancement in quality, sensorial properties and stability. Figure 1: preparation of the different tofu samples Table 1: different tofu samples inoculated with various spoilage microorganisms MATERIALS AND METHODS Isolation and identification of spoilage strains: Five different contaminated tofu samples were used for the isolation of the typical spoilage flora. Genomic DNA was extracted from pure cultures. The bacteria cultures were then identified by RAPD PCR (primer M13) and sequencing of the 16S rRNA region by BMR Genomics s.r.l (Padova, Italy). Isolation and identification of LAB strains: In order to select the most active lactic acid bacteria in terms of soy milk fermentation a screening with 9 different LAB species belonging to the collection of Food Science Department of Bologna University, was performed. Each LAB strain was inoculated in 20 ml of soymilk at a 10 8 CFU/ml level and incubated at 37°C.a Growth of the bacteria and changes in the pH of the medium were monitored at 0, 5, 17 and 24 hours. The two most performing strains (selected by best growth and acid production) were chosen for the next test; these strains were: L. casei LbCD2 and L. acidophilus LA1. Tofu preparation at laboratory scale: Three different kind of tofu were prepared at laboratory scale: 1) traditional tofu; 2) tofu fermented with L. casei e L. acidophilus and 3) tofu acidified at pH 5.5 by lactic acid addition. The preparation of the different samples is resumed in figure 1. Shelf life evaluation: The tofu samples were stored at 6°C for 42 days, testing the pH, the volatile compounds and the microbial growth after 0, 7, 28 and 42 days. The microbiological test was performed using MRS media (for LAB) and PCA for the total mesophilic count. In order to evaluate the volatile compounds 3 g of sample were used for the GC- MS/SPME analysis, stored in vials at -20°C. Set up of challenge test: The strains used for the challenge test were selected on the basis of their ability to resist at low pH and on the basis of their species. The resistance to low pH was developed in PCA for all the bacteria isolated or in MRS media (Oxoid, England) for lactic acid bacteria. The pH tested were 4, 5 and 6. The pH values were reached by lactic acid addiction. The strains selected were: Leuconostoc lactis, Pseudomonas fragii and Enterococcus faecium. Overnight growing cultures (10 3 CFU/ml) were inoculated in tofu samples, as shown in table 1. These samples were stored at 10°C and the bacterial growth was monitored at 0, 3, 10 and 20 days. Analysis of volatile compounds: The analysis of volatile compounds in fermented soy milk (3g vial) was developed by GC-MS/SPME in agreement with the method described by Serrazanetti et al., 2011. Data preprocessing by XCMS: All the GC–MS raw files were converted to netCDF format via Chemstation (Agilent Technologies, USA) and subsequently processed by the XCMS toolbox (http://metlin.scripps.edu/download/). XCMS software allows an automatic and simultaneous retention time alignment, matched filtration, peak detection, and peak matching. The resulting table containing information such as peak index (retention time-m/z pair) and normalized peak area was exported into R (www.r-project.org) for subsequent statistical analyses. Electronic nose: The analyses were performed with a portable device (Pen 2 Airsense Analytics), composed by 10 analytic sensor; each one of them is useful for different kind of volatile molecules. For every sample three repetition of the analysis were performed. On every analysis the response of the sensors were monitored each second for an overall time of 95 second. Results are expressed by comparing the signal of every sensor at every time with the minimum signal level. Signal evaluation has followed the procedure showed by Sado Kamden et al., (2007) in order to found out which are the most indicative signal for the evaluation of the differences between the different samples. Statistical analysis: The most significant type of signal was determined by ANCOVA (PLS), in order to discriminate the samples obtained by electronic nose. The first six most representative sensors were then used to create "Mixed Feature" (MF). The MF was used for the next statistic speculations. Construction of heat map: A heat map, visualizing metabolites concentration and electronic nose MF, was then obtained in which values are represented by cells colored according to the Z-scores (Z= (observed value – mean)/standard deviation). On the basis of the results obtained, L. casei LbCD2 and L. acidophilus LA1 were selected for the next studies. The two strains alone or in combination were inoculated in soymilk and a new tofu, with milk fermented by LAB was produced. This product was compared with traditional tofu and tofu produced with acidified milk (by the addition of lactic acid till pH 5.5). The samples obtained by soy milk fermentation resulted more stable in thermal abuse conditions during 42 days (Figure 2). Fermentation of Soy milk – Shelf life evaluation A preliminary screening on different LAB species was set up inoculating the strains (10 2 CFU/ml) in tubes containing soymilk. The soymilk inoculated was incubated at 37°C. The decrease of pH and the strains growth ability were followed at 0, 5, 17 and 24 hrs. The strains that grown faster were L. casei LbCD2 and L. acidophilus LA1. In 17 hours the cells concentration, of the both strains, was about 10 10 CFU/ml of soymilk. Moreover, when the both strains were used as fermenting agents, the pH of the system decreased constantly after the inoculum, and a pH around 5.1-5.5 was reached in 17 hours (data not shown). 0 1 2 3 4 5 6 7 0 7 28 42 Spilage microbial charge (logUFC/g) Time (days) TOFU1 (LbCD2) TOFU2 (LA1) TOFU3 (LbCD2+LA1) Figure 2: shelf life evaluation (plate count in PCA media) of the different tofu samples produced in terms of spoilage microbial evolution Standard L. casei LbCD2 L. acidophilus LA1 L. casei LbCD2 + L. acidophilus LA1 Lactic acid Volatile compounds Latte TQ TOFU T0 TOFU4 T7 TOFU4 T28 TOFU4 T42 TOFU1 T7 TOFU1 T28 TOFU1 T42 TOFU2 T7 TOFU2 T28 TOFU2 T42 TOFU3 T7 TOFU3 T28 TOFU3 T42 TOFU5 T7 TOFU5 T28 TOFU5 T42 HEXANAL 28.1 15.37 36.33 3.38 1.86 11.43 0.07 8.66 12.61 12.15 14.4 10.3 0.33 14.1 44.99 18.94 31.82 2,4- NONADIENAL 5.17 11.51 16.51 13.36 15.23 0.67 0.13 12.54 12.05 9.75 20.07 15.92 14.63 16.62 16.51 10.22 8.18 1-HEXANOL 20.1 32.76 22.57 30.31 31.75 10.4 0.2 31.85 31.94 27.37 27.17 33.36 31.71 30.58 22.56 29.72 23.65 ACETIC ACID 0.24 10.91 5.16 6.75 6.79 0.61 0.33 10.89 14.53 12.3 16.79 15.92 15.34 15.3 5.6 7.23 4.55 Table 2: Volatile compounds (mg/l) produced in the different tofu samples Tofu samples obtained following LAB fermentation, after 20 days, can be clearly distinguished from the standard sample, in terms of volatile compounds production (table 2 and figure 3). That changes are influenced by the process and influencing the stability and the quality of the final products. Figure 3: volatile compounds profiles of the sample obtained by lactic acid bacteria soy milk fermentation (A) and by spontaneous fermentation (B) 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 50000 100000 150000 200000 250000 300000 350000 400000 450000 Time--> Abundance TIC: 1FF.D 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 50000 100000 150000 200000 250000 300000 350000 400000 450000 Time--> Abundance TIC: 2FF.D A B Challenge test In order to confirm the microbial stability of tofu obtained by LAB soy milk fermentation, contaminating microorganisms (L. lactis, P. fragile and E. faecium) were inoculated in the different samples. The soy milk was fermented for 15 hours at 22°C until reaching the limit of pH 5.5. The samples were inoculated by spoilage microorganism before pasteurization (75 ° C for 10 minutes: VP 70 = 31.6 minutes). The samples were stored at 10°C in order to accelerate any degradation processes. 0 1 2 3 4 5 6 T3 T10 T18 logUFC/g in PCA media Time (days) Leuconostoc Enterococcus Pseudomonas not inoculated Leuconostoc Enterococcus Pseudomonas not inoculated Figure 4: spoilage microorganism evolution in the samples obtained by LAB soy milk fermentation or in standard samples. Standard Process Tofu obtained by soy milk LAB fermentation -5 -4 -3 -2 -1 0 1 2 3 4 5 -7 -6 -5 -4 -3 -2 -1 0 1 2 3 4 5 6 7 t[2] t[1] 15XCMS.M3 (PLS) R2X[1] = 0.5804 R2X[2] = 0.304908 Ellipse: Hotelling T2 (0.95) LaLc_Ll LaLc_Ps LaLc_T2 LaLc_n.c Conv_Ll Conv_Ps Conv_T2 Conv _n.c. pH 5.5_Ll pH 5.5_Ps pH 5.5_T2 pH 5.5_n.c SIMCA-P+ 11.5 - 11/20/2010 3:46:55 PM Figure 5: characterization of tofu samples, inoculated with Leuconostoc lactis (Ll), Enterococcus faecium(T2) and Pseudomonas fragi (Ps) before the thermal treatment, after 20 days of storage. The samples were monitored during the shelf life also by GC-MS-SPME and electronic nose. The results obtained with both techniques were elaborated by a combination of multivariate statistical methods such as PCA and PLS. In general, the samples stored at 3 and 10 days are not distinguishable either in relation to the process or in relation at the time (data not shown). However, the samples stored for 20 days, and in particular the fermented ones, clustered separately (Figure 5). The relation between the plate count analysis and the sensorial analyses (GC-MS-SPME and electronic nose) pointed out that the both techniques can distinguish the process of fermented samples and also the stability of those samples during the storage time. Tofu obtained by soy milk LAB fermentation Standard Process Tofu obtained with lactic acid soy milk acidification REFERENCES Sado Kamdem S., Ndagijimana M., Vannini L., Guerzoni M. E. IJFS. Special Issue, 393-399 (2007). Ryu Y., Won B., Park H., Ghafoor K., Park J. J Sci Food Agric. 90: 843–849 (2010). Serrazanetti D.I., Ndagijimana M., Sado S.L., Corsetti A., Vogel R. F., Ehrmann M., Guerzoni M. E. Appl Environ Microbiol. 77: 2656-66 (2011).
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OBJECTIVE
The aim of this study is to improve the nutritional and sensorial quality and the shelf life of commercial tofu by lactic acid fermentation.
INTRODUCTION Tofu, a fundamental part of Asian food culture, is a traditional oriental soybean food composed principally of protein and oil (Kitawaki et al., 2009). Contemporary, its consume and popularity is growing rapidly in popularity in the West. With the intent to preserve its quality for a longer period of time, tofu is often pasteurized or packed under vacuum. It is possible that these treatments are not enough to prevent the development of spoilage or pathogen microorganisms. Tofu pH is around 6.5 and Aw around 1: these characteristics make it susceptible to spoilage microorganisms affecting shelf-life. Fermentation of soy-milk by probiotics leads to a development of a new more stable functional food with bioactive components. In this study a new way to produce tofu with soy-milk fermentation by probiotic lactic acid bacteria (Lactobacillus casei and Lactobacillus acidophilus) has been developed, in order to prevent undesired microbial growth, improve stability and nutritional quality of the product. Moreover, to confirm the stability of the tofu obtained with fermented soymilk, a challenge test, with microorganisms isolated from contaminated tofu, has been set up.
Diana I. Serrazanetti*1,2, Maurice Ndagijimana3, Claudio Miserocchi4, Eleonora Tosato4, M. Elisabetta Guerzoni1,2
1 Department of Food Science, University of Bologna, Via Fanin, 44 – 40127 Bologna, Italy. 2 Inter-departmental Center of Industrial Agri-Food Research (CIRI Agroalimentare), P.zza Goidanich 60, 47521, Cesena, Italy.
3 Department of Agricultural Food & Nutritional Science 410, Agriculture/Forestry Centre University of Alberta, Edmonton, AB, Canada. 4 Con.Bio. s.r.l. Via Del Salice, 42 - 47822 Santarcangelo di Romagna (RN), Italy
CONCLUSIONS • The lactic acid fermentation lead to a new tofu (or new ingredient) whit an increased shelf life. • The product obtained can be simply distinguished by the traditional product from the sensorial and analytical point of
view. • The lactic acid fermentation can be also interesting for the improved nutritional effects on soy products (conversion of
isoflavones in aglicones and antioxidant compounds production) (Ruy et al., 2010).
Innovative methods of tofu production by soy milk fermentation. Enhancement in quality, sensorial properties and stability.
Figure 1: preparation of the different tofu samples
Table 1: different tofu samples inoculated with various spoilage microorganisms
MATERIALS AND METHODS Isolation and identification of spoilage strains: Five different contaminated tofu samples were used for the isolation of the typical spoilage flora. Genomic DNA was extracted from pure cultures. The bacteria cultures were then identified by RAPD PCR (primer M13) and sequencing of the 16S rRNA region by BMR Genomics s.r.l (Padova, Italy). Isolation and identification of LAB strains: In order to select the most active lactic acid bacteria in terms of soy milk fermentation a screening with 9 different LAB species belonging to the collection of Food Science Department of Bologna University, was performed. Each LAB strain was inoculated in 20 ml of soymilk at a 108 CFU/ml level and incubated at 37°C.a Growth of the bacteria and changes in the pH of the medium were monitored at 0, 5, 17 and 24 hours. The two most performing strains (selected by best growth and acid production) were chosen for the next test; these strains were: L. casei LbCD2 and L. acidophilus LA1. Tofu preparation at laboratory scale: Three different kind of tofu were prepared at laboratory scale: 1) traditional tofu; 2) tofu fermented with L. casei e L. acidophilus and 3) tofu acidified at pH 5.5 by lactic acid addition. The preparation of the different samples is resumed in figure 1. Shelf life evaluation: The tofu samples were stored at 6°C for 42 days, testing the pH, the volatile compounds and the microbial growth after 0, 7, 28 and 42 days. The microbiological test was performed using MRS media (for LAB) and PCA for the total mesophilic count. In order to evaluate the volatile compounds 3 g of sample were used for the GC-MS/SPME analysis, stored in vials at -20°C. Set up of challenge test: The strains used for the challenge test were selected on the basis of their ability to resist at low pH and on the basis of their species. The resistance to low pH was developed in PCA for all the bacteria isolated or in MRS media (Oxoid, England) for lactic acid bacteria. The pH tested were 4, 5 and 6. The pH values were reached by lactic acid addiction. The strains selected were: Leuconostoc lactis, Pseudomonas fragii and Enterococcus faecium. Overnight growing cultures (103 CFU/ml) were inoculated in tofu samples, as shown in table 1. These samples were stored at 10°C and the bacterial growth was monitored at 0, 3, 10 and 20 days. Analysis of volatile compounds: The analysis of volatile compounds in fermented soy milk (3g vial) was developed by GC-MS/SPME in agreement with the method described by Serrazanetti et al., 2011. Data preprocessing by XCMS: All the GC–MS raw files were converted to netCDF format via Chemstation (Agilent Technologies, USA) and subsequently processed by the XCMS toolbox (http://metlin.scripps.edu/download/). XCMS software allows an automatic and simultaneous retention time alignment, matched filtration, peak detection, and peak matching. The resulting table containing information such as peak index (retention time-m/z pair) and normalized peak area was exported into R (www.r-project.org) for subsequent statistical analyses. Electronic nose: The analyses were performed with a portable device (Pen 2 Airsense Analytics), composed by 10 analytic sensor; each one of them is useful for different kind of volatile molecules. For every sample three repetition of the analysis were performed. On every analysis the response of the sensors were monitored each second for an overall time of 95 second. Results are expressed by comparing the signal of every sensor at every time with the minimum signal level. Signal evaluation has followed the procedure showed by Sado Kamden et al., (2007) in order to found out which are the most indicative signal for the evaluation of the differences between the different samples. Statistical analysis: The most significant type of signal was determined by ANCOVA (PLS), in order to discriminate the samples obtained by electronic nose. The first six most representative sensors were then used to create "Mixed Feature" (MF). The MF was used for the next statistic speculations. Construction of heat map: A heat map, visualizing metabolites concentration and electronic nose MF, was then obtained in which values are represented by cells colored according to the Z-scores (Z= (observed value – mean)/standard deviation).
On the basis of the results obtained, L. casei LbCD2 and L. acidophilus LA1 were selected for the next studies. The two strains alone or in combination were inoculated in soymilk and a new tofu, with milk fermented by LAB was produced. This product was compared with traditional tofu and tofu produced with acidified milk (by the addition of lactic acid till pH 5.5). The samples obtained by soy milk fermentation resulted more stable in thermal abuse conditions during 42 days (Figure 2).
Fermentation of Soy milk – Shelf life evaluation A preliminary screening on different LAB species was set up inoculating the strains (102 CFU/ml) in tubes containing soymilk. The soymilk inoculated was incubated at 37°C. The decrease of pH and the strains growth ability were followed at 0, 5, 17 and 24 hrs. The strains that grown faster were L. casei LbCD2 and L. acidophilus LA1. In 17 hours the cells concentration, of the both strains, was about 1010 CFU/ml of soymilk. Moreover, when the both strains were used as fermenting agents, the pH of the system decreased constantly after the inoculum, and a pH around 5.1-5.5 was reached in 17 hours (data not shown).
0
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0 7 28 42
Spilage m
icrobial cha
rge (lo
gUFC/g)
Time (days)
TOFU1 (LbCD2)
TOFU2 (LA1)
TOFU3 (LbCD2+LA1)
Figure 2: shelf life evaluation (plate count in PCA media) of the different tofu samples produced in terms of spoilage microbial evolution
Standard L. casei LbCD2 L. acidophilus LA1 L. casei LbCD2 + L. acidophilus LA1 Lactic acid
Volatile compounds Latte TQ TOFU T0 TOFU4 T7 TOFU4 T28 TOFU4 T42 TOFU1 T7 TOFU1 T28 TOFU1 T42 TOFU2 T7 TOFU2 T28 TOFU2 T42 TOFU3 T7 TOFU3 T28 TOFU3 T42 TOFU5 T7 TOFU5 T28 TOFU5 T42
HEXANAL 28.1 15.37 36.33 3.38 1.86 11.43 0.07 8.66 12.61 12.15 14.4 10.3 0.33 14.1 44.99 18.94 31.82
2,4-NONADIENAL 5.17 11.51 16.51 13.36 15.23 0.67 0.13 12.54 12.05 9.75 20.07 15.92 14.63 16.62 16.51 10.22 8.18
1-HEXANOL 20.1 32.76 22.57 30.31 31.75 10.4 0.2 31.85 31.94 27.37 27.17 33.36 31.71 30.58 22.56 29.72 23.65
ACETIC ACID 0.24 10.91 5.16 6.75 6.79 0.61 0.33 10.89 14.53 12.3 16.79 15.92 15.34 15.3 5.6 7.23 4.55
Table 2: Volatile compounds (mg/l) produced in the different tofu samples
Tofu samples obtained following LAB fermentation, after 20 days, can be clearly distinguished from the standard sample, in terms of volatile compounds production (table 2 and figure 3). That changes are influenced by the process and influencing the stability and the quality of the final products. Figure 3: volatile compounds profiles of the sample obtained by lactic acid
bacteria soy milk fermentation (A) and by spontaneous fermentation (B)
4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00
50000
100000
150000
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250000
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350000
400000
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Time-->
Abundance
TIC: 1FF.D
6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00
50000
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150000
200000
250000
300000
350000
400000
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Time-->
Abundance
TIC: 2FF.D
!
A
B
Challenge test In order to confirm the microbial stability of tofu obtained by LAB soy milk fermentation, contaminating microorganisms (L. lactis, P. fragile and E. faecium) were inoculated in the different samples. The soy milk was fermented for 15 hours at 22°C until reaching the limit of pH 5.5. The samples were inoculated by spoilage microorganism before pasteurization (75 ° C for 10 minutes: VP70 = 31.6 minutes). The samples were stored at 10°C in order to accelerate any degradation processes.
0
1
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3
4
5
6
T3 T10 T18
logU
FC/g in PCA
med
ia
Time (days)
Leuconostoc Enterococcus Pseudomonas not inoculated Leuconostoc Enterococcus Pseudomonas not inoculated
Figure 4: spoilage microorganism evolution in the samples obtained by LAB soy milk fermentation or in standard samples.
Standard Process
Tofu obtained by soy milk LAB fermentation
-5
-4
-3
-2
-1
0
1
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-7 -6 -5 -4 -3 -2 -1 0 1 2 3 4 5 6 7
t[2]
t[1]
15XCMS.M3 (PLS)t[Comp. 1]/t[Comp. 2]Colored according to Obs ID (class)
R2X[1] = 0.5804 R2X[2] = 0.304908 Ellipse: Hotelling T2 (0.95)
LaLc_LlLaLc_Ps
LaLc_T2LaLc_n.c
Conv_Ll
Conv_Ps
Conv_T2
Conv _n.c.pH 5.5_LlpH 5.5_PspH 5.5_T2pH 5.5_n.c
SIMCA-P+ 11.5 - 11/20/2010 3:46:55 PM!
Figure 5: characterization of tofu samples, inoculated with Leuconostoc lactis (Ll), Enterococcus faecium(T2) and Pseudomonas fragi (Ps) before the thermal treatment, after 20 days of storage.
The samples were monitored during the shelf life also by GC-MS-SPME and electronic nose. The results obtained with both techniques were elaborated by a combination of multivariate statistical methods such as PCA and PLS. In general, the samples stored at 3 and 10 days are not distinguishable either in relation to the process or in relation at the time (data not shown). However, the samples stored for 20 days, and in particular the fermented ones, clustered separately (Figure 5). The relation between the plate count analysis and the sensorial analyses (GC-MS-SPME and electronic nose) pointed out that the both techniques can distinguish the process of fermented samples and also the stability of those samples during the storage time.
Tofu obtained by soy milk LAB fermentation
Standard Process
Tofu obtained with lactic acid soy milk acidification
REFERENCES Sado Kamdem S., Ndagijimana M., Vannini L., Guerzoni M. E. IJFS. Special Issue, 393-399 (2007). Ryu Y., Won B., Park H., Ghafoor K., Park J. J Sci Food Agric. 90: 843–849 (2010). Serrazanetti D.I., Ndagijimana M., Sado S.L., Corsetti A., Vogel R. F., Ehrmann M., Guerzoni M. E. Appl Environ Microbiol. 77: 2656-66 (2011).
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