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JOURNAL OF CULTURE COLLECTIONS=

Volume 5, 2006-2007, pp. 46-57=

 

 

 

CHARACTERIZATION OF LACTIC ACID BACTERIA
FROM TRADITIONAL THAI FERMENTED SAUSAGES

 =

Chantaraporn Phalakornk= ule1* and Somboon Tanasupawat2

 

1= Department of Chemical Engineering, Faculty of Engineering, King Mongkut’s Insti= tute of Technology,
North Bangkok 10800, Thailand;
2Department of Microbiology, Faculty of Pharmaceutical Sciences, Chulalongk= orn University, Bangkok 10330, Thailand

*Corresponding author, e-mail: cpk@kmitnb.ac.th, cphalak21@yahoo.com

 

 

Summary

Lactic acid bacteri= a from traditional Thai fermented sausages were characterized. The fermented sausa= ges were mainly produced from minced pork/beef or pork/beef liver. Sixty five s= trains were isolated from 12 samples collected from the central and northeast= ern parts of Thailand. The strains were identified by conventional morphological, cultural, physiological and biochemical tests as well as by 16S rDNA sequence analysi= s. The isolates were identified as = Weissella cibaria/kimchii (5), W. confusa (3), Pediococcus pentosaceus (20), <= span style=3D'mso-bidi-font-style:italic'>P. acidilactici (2), Lactobacillus fermentum (3), <= span style=3D'mso-bidi-font-style:italic'>L. brevis (4), L. farciminis (4), L. plantarum (23), and L. sakei (1). Some of these species have not been previously isolated f= rom Thai fermented sausages. The inhibition tests against Bacillus cereus and Staphylococcus aureus showed that 5 isolates could inhibit the growth of B. cereus and 2 of them could a= lso inhibit S. aureus. The is= olates were identified as W. confusa (1), P. acidilactici (1),= L. plantarum (3). Three st= rains identified as L. plantarum and one as Weissella spp. cou= ld produce diacetyl.  =

Key words: fermented sausage, lactic acid bacteria, Weissella, Lactob= acillus, Pediococcus, 16S DNA, dia= cetyl, bacteriocin.

&= nbsp;

&= nbsp;

Introduction

 “Sai-krork-prieo” is a traditional Thai fermented sausage, consumed all around the country and produced from minced pork, garlic, pepper, spices, salt and sugar. “Mum” is a product similar to “Sai-krork-prieo”, but can be made from beef as well as pork, and is popular only in the Northeast= ern of Thailand.

Lactic acid bacteria (LAB) play an important role in the ripening process of raw fermented sausages. However, = the production of traditional fermented sausages in Thailand has utilized the nat= urally occurring LAB, resulting in various and inconstant products. Natural fermentations could vary from the simple to the complex ones. In general, e= ach fermentation takes place under conditions that the producers have found to = be favorable for the appropriate growth and action of micro-organisms. Alterna= tive to the natural fermentation, the use of well-studied starter cultures would result in more constant and safe food products.

The aim of this study was the isola= tion and identification of LAB from traditional fermented sausages in Thailand. In addition, their capability of producing metabolic compounds with antimicrob= ial property, i.e., diacetyl and bacteriocins, is reported. The strains found in this study have a potential use in the establishing of the so-called “functional foods” [12, 17].

 

Materials and methods

Sample collection, bacterial cell counts, and isolation method. Twelve fermented sausage samples of various brands were collec= ted from factories and local markets. The pH of the samples was represented by = the pH of the suspension of a 5-gram portion in 10 ml deionized water. = The cell numbers were counted by a plating method on De Man, Rogasa and Sharpe = agar (MRS agar) [3] at 30 oC after a 3- to 5-day incubation. Pure cultures were obtained by streaking cultured cells on MRS agar plates with 0.2 % CaCO3.

Morphological and cultural characteristics. Cell form, cell size, cell arrangement, and colony appearance = were examined on cells grown on a half strength MRS (MRSH) agar incubated for 3 days. Hucker-Conn modification [8] was used for gram stain. Spore formation= was examined in gram-stained specimens. Motility was detected by the appearance= of stab cultures in soft agar [30].

Biochemical and physiological characteristics. Catalase, nitrate reduction, hydrolysis of esculin, arginine, = slime formation and re-actions in litmus milk were investigated as described by Tanasupawat et al. [20, 2= 3]. Catalase activity was detected on cel= ls grown on MRSH agar. Nitrate reduction was tested after incubation of cells for 7 = days in a medium com-posed of 1.0 g KNO3, 3.0 g yeast extra= ct, 5.0 g peptone, 0.2 g beef extract, 5.0 g NaCl, 0.25 g T= ween 80, 1.0 g agar, and 1000 ml deionized water, adjusted to pH 6.8. Growt= h at different temperature (30, 45, and 50 oC), at different starting pH (3.5, 4.0, 4.5, 8.0, 8.5 and 9.6), and at different concentrati= ons of NaCl (4, 6, 8, and 10 %) were tested by using MRSH broth. The production of gas from D-glucose was examined by using MRS broth with a Durham tube. Acid production from carbohydrates was deter-mined as described in Tanasupawat <= span style=3D'mso-bidi-font-style:italic'>et al. [23] by the use of a bas= al medium of GYPB broth with the omission of glucose. The GYPB medium contained 10.0 g glucose, 5.0 g yeast ex-tract, 5.0 g peptone, 2.0&nbs= p;g beef extract, 2.0 g sodium acetate, 0.25 g Tween 80, 200 mg = MgSO4= ×7H2O, 10 mg MnSO4×= 4H2O, 10 mg FeSO4×= 7H2O, 5.0 g NaCl, and 1000 ml deioni-zed water, adjusted to pH 6.8. The acid produced in 3 ml broth was titrated with 0.1 N NaOH.

Peptidoglycan type of the cell wall. Me-so-diaminopimelic acid (DAP) in the cell wall was detected by hydrolysis= of 3 mg dried cells grown in GYPB broth. Hydrolysis was perform-ed with 1 = ;ml 6 N HCl at 100 oC for 18 h, and the hydrolysate was applied= on cellulose TLC plate (Merck no. 5577). The TLC plate was developed with the solvent system of methanol-pyridine-12 N HCl-water (80: 10: 1.5: 17.5) (v/v) [11]. Spots were visualized by spraying with 0.5 % ninhydrin solution = in n-butanol followed by heating at 100 oC for a few minutes.<= /span>

Isomers of lactic acid. The strains tested were cultivated in G= YPB broth for 3 to 5 days. Lactic acid was analyzed enzymatically ac-cording to Okada et al. [15] using D= - and L-lactate dehydrogenase (Boehr= inger, Germany).=

Screening for diacetyl formation. Diacetyl f= ormation was screened by the method modified from Mattessich and Cooper [13] and Pha= lip et al. [16], which yi= elded qualitative results. Cells were grown in MMRS broth at 35 = oC for 3-5 days. Hundred ml of the MMRS broth contained 0.18 g glucose, 0.5 g yeast extract, 1.0 g peptone, 1.0 g beef extract, 0.5 g sodium acetate, 1.29 g trisodium citrate x 2H2O, 0.15 g Tween 80, 0.02 g MgSO4×= 7H2O, 0.005 g MnSO4= ×H2O, and 0.2 g K2HPO4. O= ne ml of each test solution (0.5 % creatine solution and 7.5 % <= span lang=3DEN-US style=3D'font-family:Symbol;mso-ascii-font-family:"Times New R= oman"; mso-hansi-font-family:"Times New Roman";mso-char-type:symbol;mso-symbol-fon= t-family: Symbol'>= a-naphthol in 2.5 N NaOH) was added into each 3 ml of culture m= edium. The degree of change in the culture medium color was re-corded.

Screening for bacteriocin production. The screening method was modified from Yin et al. [31]. Each LAB strain was grown on Trypticase soy agar (without glucose) with a 2.0 % yeast extract supplement (TSAYE) slant = at 37 oC for 2 days. The cultured LAB cells were streaked on T= SAYE plate. Working pathogens (Bacillus cereus TISTR 037 and Staphyloc= occus aureus TISTR 029 from Thailand Institute of Scientific and Technological Research) were cultured in nutrient agar (NA) slant at 37 oC for 2 days. The cultured pathogen cells were transferred to 0.85 % sal= ine solution to obtain a cell density of 105-106 colony-forming units (CFU) per ml. One ml of the suspended pathogen cells was mixed with 15 ml of mel= ted Trypticase soy agar (TSA) at 50 oC. The melted TSA was then overlaid on the TSAYE plate with growing LAB. The plate with the overlay was incubated anaerobically at 37 oC for 2 days. The control TS= AYE plate with a pathogen over-lay but without cultured LAB was incubated in parallel for comparison. Inhibition zones reveal-ed by colony appearance we= re recorded.

Sequencing and comparison of 16S rRNA gene. Template DNA for 16S rRNA gene amplification was prepared by the method modified from Nilsson et = al. [14]. Two loops from an overnight MRS plate culture were transferred into 100-500 µl of TE buffer (10 mM Tris-Cl, pH 7.5, and 1 mM EDTA). Samples we= re boiled for 10-15 min. Then, the debris was pelleted by centrifugation at 12000 rpm for 10 min, and 50-200 µl of the supernatant was collected. The 16S rRNA gene was amplified using the PCR method with a 1U Taq DNA polymerase (Bio-Lab Ltd., Auckland, New Zealand) and the primers UFUL (GCCTAAC= ACATGCAAGTCGA) and URUL (CGTATTACCGCGGCTGCTGG) (Great American Gene Co., California, USA). These primers target two highly conserved regions known to be variable among bacterial species [4, 2= 9]. The 16S rRNA gene was sequenced by using a BigDye v. 3.1 cycle sequencing k= it (Applied Biosystems, California, USA), according to the manufacturer’s protocol, with UFUL as primer. The 16S rRNA gene sequences determined (ca. 400-500 bases) were aligned along with the sequences of type strains obtain= -ed from the GenBank by using the program CLUSTAL X (version 1.82) [28]. Distan= ce matrices for aligned sequences were calculated by the two-parameter method = of Kimura [10]. A phylogenic tree was constructed by the neighbor-joining meth= od [18] with the program PHY-LIP (version 3.64) available at http://evolution. genetics.washington.edu/phylip.html. Confidence values of individual branch= es in the phylogenetic tree were determined by using the bootstrap analysis of Felsenstein [5] based on 1,000 samplings.

Reference strains. Weissella confusa ATCC 10881T, W. cibaria/kimchii LMG 17699T, Pediococcus pentosaceus ATCC 33316<= sup>T, P. acidilactici DSM 20284T, Lactobacillus fermentum ATCC 14931T, L. brevis ATCC 14869T, = L. farciminis ATCC 29644T, L. plantarum ATCC 14917T and L. sakei ATCC 15521T were used as reference stra= ins.

 

Results

Bacterial cell counts and sausage characteristics=

Twelve samples from Thai fermented sausages collected from the central and north-eastern parts of Thailand were studied. The te= sted samples contained 1.4 x 1011-5.5 x 1013 bacterial cells/g, and showed a pH between 4.2 and 5.0 (Table 1).

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Table 1. Bacterial cell counts and sau= sage characteristics.

Sample No.

Sausage names and province where collected

Days of fermen-tation

pH

Bacterial counts (cells/g)

Isolate No.

(65 isolates)

CP1

Sai-krork-prieo  Bangkok

4

4.2

3.7x1011

CP1-5, CP1-8, CP1-13, CP1-14, <= br> CP1-15, CP1-17, CP1-18, CP1-19,
CP1-20  

CP2

Sai-krork-prieo Pathumthani

5

4.2

2.5x1012

CP2-3A, CP2-3B, CP2-10, CP2-11,=
CP2-16 

CP3

Sai-krork-prieo Pathumthani

4

4.9

1.4x1011

CP3-1, CP3-8, CP3-9, CP3-10, CP3-11, CP3-16       

CP4

Sai-krork-prieo Pathumthani

4

4.8

1.9x1012

CP4-5, CP4-6, CP4-11, CP4-12, <= br> CP4-16, CP4-17, CP4-18      <= /p>

CP5

Sai-krork-prieo Bangkok

2

4.3

4.0x1013

CP5-2, CP5-6, CP5-9    

CP6

Mum (beef)

Chaiyaphoom

3

4.5

3.0x1013

CP6-2, CP6-3, CP6-8, CP6-10,    

CP7

Mum (beef) Chaiyaphoom

3

4.4

5.5x1013

CP7-2, CP7-3, CP7-4, CP7-5,
CP7-7, CP7-8, CP7-9, CP7-10,
CP7-12, CP7-13

CP8

Mum (beef) Konkean

4

4.6

1.5x1012

CP8-1, CP8-4       &nbs= p;  

CP10

Mum (pork) Chaiyaphoom

4

5.0

8.0x1011

CP10-2, CP10-3       

CP11

Sai-krork-prieo Chaiyaphoom

4

4.6

1.0x1013

CP11-2, CP11-3, CP11-5, CP11-7,=
CP11-8, CP11-10, CP11-11, CP11-13,
CP11-14, CP11-15      

CP12

Mum (pork) Chaiyaphoom

4

5.0

2.0x1013

CP12-4

CP13

Sai-krork-prieo Chaiyaphoom

4

4.8

8.5x1012

CP13-1, CP13-2, CP13-3, CP13-4,=
CP13-7, CP13-11

 

Morphological and cultural characteristics

All isolates were Gram-positive, non-motile, and non-sporing. = The cells of 30 sphere-shaped strains measured 0.8 to 1.0 µm in size and appeared in pairs or in tetrads (Table 2). Their colonies on MRS agar plates were circular, low convex with entire margin, and non-pigmented. The cells = of 35 rod-shaped strains measured 0.8-1.0 x 1.5-5.0 µm in cell size, and appeared singly, in pairs, or in chains (Table 3). Their colonies= on MRS agar plates were circular, low convex with entire margin, and non-pigmented. 

 

Table 2. General characteristics of group A strains.

Characteristics

A1

1 W. confusa
ATCC 10881T

2 W. kimchii
JCM 12495T

A21

3 P. pento-
saceus
ATCC 33316T

A22

3 P. acidi-
lactici
 DSM 20284T

Numb= er of strains

8

-

-

20

-

2

-

Cell= form

Cocci

Cell arrangement

In pairs or tetrads

Gas = from glucose

+

+

+

-

-

-

-

Growth at

 

 

 

 

 

 

 

45 oC

-

-

-

+

-

+

+

50 oC<= /span>

-

-

-

-

-

+

+

Isom= ers of lactic acid

DL

DL

D

DL

DL

DL

DL

Argi= nine hydrolysis

+

+

+

+

-

+

+

Escu= lin hydrolysis

+

+

+

+

+

+

+

Nitr= ate Reduction

-

ND

ND

-

-

-

-

Reaction in litmus milk

 

 

 

 

 

 

 

Acidification

- (+2)

ND

ND

-

-

-

-

Coagulation

- (+2)

ND

ND

-

-

-

-

Reduction

-

ND

ND

-

-

-

-

Growth at pH

 

 

 

 

 

 

 

3.5

- (+1)

-

-

+(-2)

ND

+(-1)

ND

4.0

+

+

W

+

+

+

+

4.5

+

+

+

+

+

+

+

8.0

+

+

+

+

+

+

+

8.5

- (+3)

-

-

+(-2)

-

+

-

9.6

-

-

-

-

-

-

-

Salt tolerance<= /p>

 

 

 

 

 

 

 

4 % NaCl

+

+

+

+

+

+

+

6 % NaCl

+

+

+

+(-1)

+

+

-

8 % NaCl

+

-

-

+(-2)

-

+

-

10 % NaCl=

-

-

-

-

-

-

-

Slime from sucrose

+

+

+

-

-

-

-

Pept= idoglycan type: DAP

-

-

-

-

-

-

-

Legend: positive (+), weakly positive (W),= negative reaction (-), not determined (ND); numbers in parenthesis indicate the numb= er of strains showing a positive or negative reaction; diaminopimelic acid (DA= P); American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganis= men und Zellkulturen (DSM), Japan Collection of Microorganisms (JCM).

1 Data from = [1, 2, 7, 9, 19, 24, 26]; 2 Data from [1]; 3 Data from [25].=

 

Table 3. General characteristics of group B strains.

Characteristics

B11

1 = L. fer-mentum ATCC 14931T

B12

2 = L. brevis ATCC 14869T

B21

2 = L. far-ciminis ATCC 29644T

B22

3 = L. plan-tarum ATCC 14917T=

B23

4 = L. sakei ATCC 15521T

Numb= er of strains

3

-

4

-

4

-

23

-

1

-

Cell= forms

Rods

Cell arrangement

Singly, in pairs or in chains

Gas = from glucose

+

+

+

+

-

-

-

-

-

+

Growth at

45 oC

50 oC<= /span>

 

+

-

 

+

ND

 

-

-

 

-

-

 

-

-

 

-

-

 

-

-

 

-

-

 

-

-

 

-

-

Isom= ers of lactic acid

DL

DL+

L(+)

DL

DL

L

L

DL

DL

DL

DL+

D(-)

Argi= nine hydrolysis

+

-

+(-1)

-

+

+

-

-

+

+

Escu= lin hydrolysis

-(+1)

-

+

-

+

+

+

+

-

+

Nitr= ate Reduction

-(+1)

ND

-

ND

-

ND

-

ND

-

ND

Reaction in litmus milk

Acidification

Coagulation

Reduction

 

-(+1)

-

-

&= nbsp;

-

-

-

 

-

-

-

&= nbsp;

-

-

-

 

+

-

-

&= nbsp;

ND

ND

ND

 

+(-5)

-(+2)

-(+1)

 

-

-

-

 

-

-

-

&= nbsp;

ND

ND

ND

Growth at pH

3.5

4.0

4.5

8.0

8.5

9.6

 

+

+

+

+

-(+1)

-

 

-

+

+

+

+

-

 

+(-1)

+

+

-(+1)

-

-

&= nbsp;

-

+

+

+

-

-

 

+(-2)

+

+

+

+(-1)

-

&= nbsp;

ND

+

+

+

+

-

 

+(-2)

+

+

+

-(+9)

-

 

+

+

+

+

+

-

 

-

-

+

+

+

-

&= nbsp;

ND

+

+

+

+

-

Salt tolerance<= /p>

4 % NaCl

6 % NaCl

8 % NaCl

10 % NaCl=

 

+

+

-(+1)

-

 

+

-

-

-

 

+

-

-

-

&= nbsp;

+

-

-

-

 

+

+

+

+

&= nbsp;

+

+

+

+

 

+

+(-3)

-(+11)

-

 

+

+

-

-

 

+

+

-

-

&= nbsp;

+

-

-

-

Slime from sucrose

-

-

-

-

-

-

-

-

-

-

Pept= idoglycan type: DAP

-

-

-

-

-

-

+

+

-

-

Legend: positive (+), weakly positive (W), negative react= ion (-), not determined (ND); numbers in parenthesis indicate the number of strains showing a positive or negative reaction; diaminopimelic acid (DAP); American Type Culture Collection (ATCC).

<= span lang=3DEN-US>1 Data from [24]; 2= Data from [23, 27]; 3 Data from [20, 24, 26]; 4 Data from [24].

&= nbsp;

Physiological and biochemical characteristics=

Isolates were divided into two major groups, Groups A and B by= cell shape. Group A consist-ed of 30 sphere-shaped isolates, which were further divided into 2 subgroups according to gas production from glucose. Subgroup= A1 produced gas from glucose (8 strains), while subgroup A2 did not (22 strain= s). All strains in subgroup A1 did not grow at 45 oC. Subgroup = A2 was further divided into 2 subgroups ac-cording to their growth at different temperatures. Subgroup A21 grew at 45 oC (20 strains); and subgroup A22 grew at 50 oC (2 strains).

Group B consisted of 35 rod-shaped isolates, which were further divided into 2 subgroups according to gas production from glucose. Sub-grou= p B1 produced gas from glucose (7 strains), while subgroup B2 did not (28 strains). Sub-group B1 was further divided into 2 subgroups accord= ing to their growth at different temperatures. Subgroup B11 grew at 45 oC (3 strains); and subgroup B12 did not grow at 45 oC (4 strains). All strains in subgroup B2 did not grow at 45 o= C. Subgroup B2 was further divided into 2 groups according to isomers of lactic acid. Subgroup B21 produced L-lactic acid from glucose (4 strains), while subgroup B22 produced DL-lactic acid (23 strains). All strains in subgroup B22 did not produce NH3 from arginine. One strain (CP8-= 1) was separated from sub-group B22 and located in subgroup B23 be-cause it hydrolyzed arginine. The distribution of isolated strains among groups and their other general characteristics are shown in Tables 2 and 3. Tables 4 a= nd 5 present the characteristics regarding the acid production from carbohydrate= s by group A and B strains, respectively.

 

Table 4. Acid production from carbo= hydrates by group A strains.

Characteristics

A1

1<= i> W. confusa<= /span> ATCC 10881<= sup>T

2 W. kimchii JCM 12495T

A21

3 P. pento-saceus
ATCC 33316T

A22

3 P. acidi-lactici
DSM 20284T

Numb= er of strains

8

-

-

20

-

2

-

D-Am= ygdalin

+(-1)

+

+

+

ND

+

ND

L-Ar= abinose

+

-

-

+(-1)

+

+(-1)

+

D-Ce= llebiose

+

+

+

+

+

+

+

D-Fr= uctose

+

+

+

+

+

+

+

D-Ga= lactose

+(-3)

+

+

+

+

+

+

D-Gl= ucose

+

+

+

+

+

+

+

Gluc= onate

-(+3)

+

+

-

ND

-

ND

Glyc= erol

-

ND

-

-

-

-

-

Inulin

-

ND

-

-(+4)

-

-

-

Lactose

-(+1)

-

-

-(+7)

-

-

-

Maltose

+

+

+

-(+2)

+

-

-

D-Mannitol

-(+1)

-

-

-

-

-

-

D-Mannose

+

+

+

+

+

+

+

D-Melibiose

-

-

-

-(+1)

+

-

-

D-Melezitose

-

-

-

-

-

-

-

a-Methyl-D-glucoside

-

ND

-

-

-

-

-

Raffinose

-

-

-

-

W

-

-

L-Rhamnose

-(+1)

-

-

+(-3)

-

+(-1)

-

D-Ribose

-(+1)

+

-

+

+

+

+

Salicin

+

+

+

+

+

+

+

D-Sorbitol

-

-

-

-

-

-

-

Sucrose

+

+

+

+(-5)

+

+

-

D-Trehalose

-

-

-

-(+8)

+

+

+

D-Xylose

+

+

+

+(-1)

W

+

+

Legend: positive (+), weakly positive (W), negative reaction (-), not determined (ND); numbers in parenthesis indicate= the number of strains showing a positive or negative reaction.

<= span lang=3DEN-US>1 Data from [1, 2, 7, 9, 19, 2= 4, 26]; 2 Data from [1]; 3 Data from [25].

 

Table 5. Acid production from carbohydrates by group B strains= .

 

B11

1 = L. fer-mentum ATCC 14931T=

B12

1&= nbsp;L. brevis ATCC 14869T

B21

2 L. farci-minis ATCC 29644T

B22

3 = L. plan-tarum ATCC 14917T=

B23

4 = L. sakei ATCC 15521T

Numb= er of strains

3

-

4

-

4

-

23

-

1

-

D-Am= ygdalin

-(+1)

-

-

-

+(-2)

+

+

ND

-

+

L-Ar= abinose

+(-1)

-

+

+

-

-

+(-2)

+

+

+

D-Ce= llebiose

-(+1)

-

-

-

+

+

+

+

W

+

D-Fr= uctose

+

+

+

+

+

+

+

+

+

+

D-Ga= lactose

+

-

-(+2)

-

+

+

+

+

+

ND

D-Gl= ucose

+

+

+

+

+

+

+

+

+

+

Gluc= onate

-

-

-

-

-

-

+(-11)

+

W

+

Glyc= erol

-

-

-

-

-

-

-

-

W

ND

Inulin

-(+1)

ND

-

ND

-(+1)

ND

+(-11)

ND

-

ND

Lactose

+(-1)

+

-

-

+

+

+(-1)

+

+

ND

Maltose

+

-

+

-

+(-2)

+

+

+

-

ND

D-Mannitol

-(+1)

-

-

-

-

-

+

+

-

-

D-Mannose

+(-1)

-

-

-

+

+

+

+

+

ND

D-Melibiose

+

-

-

-

-

-

+(-1)

+

+

+

D-Melezitose

-(+1)

-

-

-

-

ND

+(-2)

+

-

-

a-Methyl-D-glucoside

-

-

+

-

-

-

-(+7)

ND

-

ND

Raffinose

+

-

-

-

-

-

+(-1)

+

-

-

L-Rhamnose

-(+1)

-

-

-

-

-

-(+11)

-

-

ND

D-Ribose

+

+

+

-

-

-

+

+

+

+

Salicin

-(+1)

ND

-

ND

+

+

+

+

W

 

D-Sorbitol

-(+1)

-

-

-

-

-

+(-4)

+

-

-

Sucrose

+

+