BACILLUS | Detection by Classical Cultural Techniques

I. Jenson , in Encyclopedia of Food Microbiology (Second Edition), 2014

Appendix: Formulations

Diluents/solutions
Butterfield's phosphate
Stock solution
Potassium dihydrogen phosphate 34.0   g
Distilled water 500   ml
Adjust pH to 7.2 with approximately 175   ml of 1   mol   l–1 NaOH
Adjust final volume with distilled water to 1000   ml store refrigerated
Diluent
Stock solution 1.25   ml
Distilled water to 1000   ml
Dispense and sterilize by autoclaving at 121   °C for 15   min
Peptone diluent
Bacteriological peptone 1.0   g
Distilled water to 1000   ml
Dispense and sterilize by autoclaving at 121   °C for 15   min
Cream diluent
Gum tragacanth 1.0   g
Gum arabic 1.0   g
Water 100   ml
Autoclave for 20   min at 121   °C
Tetrazolium salts
2,3,5-Triphenyl-tetrazolium chloride 10.0   g
Water to 100   ml
Sterilize by membrane filtration through a 0.2   μm filter
Nitrite reagents
Reagent A
Sulfanilic acid 8.0   g
5   mol   l–1 Acetic acid 1000   ml
Reagent B
α-naphthol 2.5   g
5   mol   l–1 Acetic acid 1000   ml
Holbrook and Anderson stain
The following solutions are required:
5%   w/v malachite green
0.3% Sudan black B in 70% ethanol
Xylol
0.5%   w/v safranin
Toxin crystal stain
0.5   g basic fuchsin dissolved in 20   ml ethanol then made up to 100   ml with water
Media for enumeration
Mannitol–egg yolk–polymyxin (MYP) agar
Beef extract 1.0   g
Peptone 10.0   g
d-Mannitol 10.0   g
Sodium chloride 10.0   g
Phenol red 0.025   g
Agar 12–18   g
Water 940   ml
Adjust pH so that it will be 7.1   ±   0.2 at 25   °C after autoclaving at 121   °C for 15   min; add 10   ml of filter-sterilized polymyxin B sulfate solution (10   000 units ml−1) and 50   ml of 50% egg yolk emulsion per 940   ml of the basal agar
Polymyxin–egg yolk–mannitol–bromothymol blue agar (PEMBA)
Tryptone 1.0   g
d-Mannitol 10.0   g
Sodium pyruvate 10.0   g
Magnesium sulfate heptahydrate 0.1   g
Sodium chloride 2.0   g
Disodium hydrogen phosphate 2.5   g
Bromothymol blue 0.12   g
Agar 12–18   g
Water 940   ml
Adjust pH so that it will be 7.2   ±   0.2 at 25   °C after autoclaving at 121   °C for 15   min; add 10   ml of filter-sterilized polymyxin B sulfate solution (1000   units   ml−1) and 50   ml of 50% egg yolk emulsion per 940   ml of the basal agar
Tryptone–soy–polymyxin broth
Tryptone 34.0   g
Soya peptone 6.0   g
Sodium chloride 10.0   g
Glucose 5.0   g
Dipotassium hydrogen phosphate 5.0   g
Water 1000   ml
Adjust pH so that it will be 7.3   ±   0.2 at 25   °C after autoclaving at 121   °C for 15   min; add 1   ml of filter-sterilized polymyxin B sulfate solution (1000   units   ml−1) per 100   ml broth
Dextrose–tryptone agar (DTA)
Tryptone 10.0   g
Dextrose 10.0   g
Agar 12–18   g
Bromocresol purple 0.04   g
Water 1000   ml
Adjust pH so that it will be 6.7   ±   0.2 at 25   °C after autoclaving at 121   °C for 15   min
Thermoacidurans agar (TAA)
Yeast extract 5.0   g
Proteose peptone 5.0   g
Dextrose 5.0   g
Dipotassium phosphate 4.0   g
Agar 20.0   g
Adjust pH so that it will be 5.0   ±   0.2 after autoclaving at 121   °C for 15   min
Tryptone–glucose extract agar (TGE)
Beef extract 3.0   g
Tryptone 5.0   g
Dextrose 1.0   g
Agar 15.0   g
Adjust pH so that will be 7.0   ±   0.2 after autoclaving at 121   °C for 15   min
Bacillus acidoterrestris medium
Basal medium
CaCl2·2H2O 0.25   g
MgSO4·7H2O 0.5   g
(NH4)SO2 0.2   g
KH2PO4 3.0   g
Yeast extract 1.0   g
Glucose 5.0   g
Trace element solution 1.0   ml
Distilled water 1.0   l
Adjust to pH 4.00; for agar the liquid medium is made up at twice the concentration and mixed with an equal volume of agar (15–20   g agar per liter) after autoclaving
Trace element solution:
CaCl2·2H2O 0.66   g
ZnSO4·7H2O 0.18   g
CuSO4·5H2O 0.16   g
MnSO4·4H2O 0.15   g
CoCl2·6H2O 0.18   g
H3BO3 0.10   g
Na2MoO4·2H2O 0.30   g
Distilled water 1.0   l
Media for confirmation
Phenol red glucose broth
Proteose peptone no. 3 10.0   g
Beef extract 1.0   g
Sodium chloride 5.0   g
Phenol red 0.018   g
Dextrose 5.0   g
Water to 1   l
Dispense in 3   ml quantities in small test tubes; autoclave for 10   min at 121   °C; final pH should be 7.4   ±   0.1
Nitrate broth
Beef extract 3.0   g
Peptone 5.0   g
Potassium nitrate 1.0   g
Distilled water to 1   l
Adjust pH to 7.0   ±   0.1 and dispense 5   ml quantities into small test tubes; autoclave 15   min at 121   °C
Modified VP medium
Proteose peptone 7.0   g
Dextrose 5.0   g
Sodium chloride 5.0   g
Water to 1000   ml
Adjust to give a pH of 6.5   ±   0.1 after autoclaving and dispense 5   ml quantities into small tubes; autoclave for 10   min at 121   °C
Tyrosine agar
Prepare nutrient agar and after autoclaving, add 10   ml of water containing 0.5   g of l-tyrosine (sterilized by autoclaving at 121   °C for 15   min) to each 100   ml of nutrient agar; dispense into slopes in sterile bottles; the tyrosine will not dissolve and must be evenly suspended throughout the agar
Nutrient broth with lysozyme
Dissolve 0.1   g lysozyme hydrochloride in 100   ml water and sterilize through a 0.2   μm membrane filter; add 1   ml of this solution to 99   ml nutrient broth; dispense 2.5   ml volumes into small sterile tubes
Nutrient broth/agar
Beef extract 3.0   g
Peptone 5.0   g
Agar (if required) 15.0   g
Water to 1000   ml
Adjust pH to give 6.8   ±   0.1 after autoclaving at 121   °C for 15   min
BC motility medium
Trypticase 10.0   g
Yeast extract 2.5   g
Dextrose 2.5   g
Disodium hydrogen phosphate 2.5   g
Agar 3.0   g
Water to 1000   ml
Adjust pH to give 7.4   ±   0.2 after autoclaving; dispense into tubes and autoclave at 121   °C for 10   min
Sheep blood agar
Trypticase 15.0   g
Phytone peptone 5.0   g
Sodium chloride 5.0   g
Agar 15.0   g
Water to 1000   ml
Adjust pH to give 7.0   ±   0.2 after autoclaving; autoclave at 121   °C for 15   min; cool to 48   °C and add 5   ml sterile defibrinated sheep blood per 100   ml medium and dispense into Petri dishes

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AEROMONAS | Detection by Cultural and Modern Techniques

B. Austin , in Encyclopedia of Food Microbiology (Second Edition), 2014

Detection by Culturing

Commonly Used Media

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Aeromonas (Ryan's) agar: 0.2% (w/v) l-arginine hydrochloride, 0.3% (w/v) bile salts no. 3, 0.08% (w/v) ferric ammonium citrate, 0.25% (w/v) inositol, 0.15% (w/v) lactose, 0.35% (w/v) l-lysine hydrochloride, 0.5% (w/v) proteose peptone, 0.5% (w/v) sodium chloride, 1.067% (w/v) sodium thiosulfate, 0.3% (w/v) sorbose, 0.375% (w/v) xylose, 0.3% (w/v) yeast extract, 1.25% (w/v) agar, 0.004% (w/v) bromthymol blue, 0.004% (w/v) thymol blue, 5   mg   l−1 ampicillin; pH 8.0; dissolve by boiling; autoclaving is not required. Aeromonas forms dark-green colonies of 0.5–1.5   mm in diameter with dark centers.

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Alkaline peptone water (APW): 1% (w/v) peptone, 1% (w/v) sodium chloride; pH 8.5–9 (typically at pH 8.5).

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Ampicillin–dextrin agar (ADA): 1% (w/v) dextrin, 0.01% (w/v) ferric chloride hexahydrate, 0.02% (w/v) magnesium sulfate heptahydrate, 0.2% (w/v) potassium chloride, 0.3% (w/v) sodium chloride, 0.5% (w/v) tryptose, 0.2% (w/v) yeast extract, 1.5% (w/v) agar, 0.004% (w/v) bromthymol blue, 10   mg   l−1 ampicillin, 100   mg   l−1 sodium deoxycholate; pH 8.0. Aeromonas spp. develop as yellow, circular, convex colonies.

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Bile salts–brilliant green agar (BBG): 1% (w/v) proteose peptone, 0.5% (w/v) Lab Lemco beef extract, 0.5% (w/v) sodium chloride, 0.85% (w/v) bile salts no. 3, 1.5% (w/v) agar, 0.000033% (w/v) brilliant green, 0.0025% (w/v) neutral red; pH 7.2; dissolve by heating; autoclaving is not required. Aeromonas produces whitish colonies on this medium.

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Bile salts–brilliant green–starch agar (BBGS): 1% (w/v) proteose peptone, 0.5% (w/v) Lab Lemco beef extract, 0.5% (w/v) sodium chloride, 0.5% (w/v) bile salts, 1% (w/v) soluble starch, 1.5% (w/v) agar, 0.005% brilliant green; pH 7.2; dissolve by heating; autoclaving is not required. After flooding with Lugol's iodine, putative Aeromonas may be visualized by the presence of clearing (indicative of starch degradation) around the colonies.

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Meso-inositol–xylose agar (MIX): 0.01% (w/v) ammonium ferric citrate, 0.2% (w/v) potassium chloride, 0.3% (w/v) sodium chloride, 0.02% (w/v) magnesium sulfate heptahydrate, 1% (w/v) meso-inositol, 0.3% (w/v) yeast extract, 0.15% (w/v) bile salts no. 3, 0.5% (w/v) xylose, 1.5% (w/v) agar, 0.0005% (w/v) bromthymol blue, 20   mg   l−1 ampicillin; pH 7.2. Aeromonas produces convex, circular blue–green colonies.

○

Modified bile salts irgasan brilliant green agar (mBIBG): 0.5% (w/v) meat extract, 0.5% (w/v) proteose peptone, 1% (w/v) soluble starch, 0.58% (w/v) bile salts no. 3, 0.544% (w/v) sodium thiosulfate, 0.0005% (w/v) irgasan, 0.0005% (w/v) brilliant green, 0.0025% (w/v) neutral red, 1.15% (w/v) agar; pH 8.7. Aeromonas develop as purple colonies after incubation at 37   °C for 24   h.

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Peptone–beef extract–glycogen agar (PBG): 1% (w/v) beef extract, 0.5% (w/v) glucose, 1% (w/v) peptone, 0.5% (w/v) sodium chloride, 0.004% (w/v) bromthymol blue, 1.5% (w/v) agar, and 2% (w/v) agar for overlay. Presumptive Aeromonas appear as yellow colonies with yellow haloes in the otherwise green medium. Ellipsoidal colonies may be seen if they are buried in the medium.

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Pril ampicillin dextrin ethanol agar (PADE): 1% (w/v) tryptose, 0.2% (w/v) yeast extract, 1.5% (w/v) dextrin, 0.02% (w/v) Pril, 0.02% (w/v) MgSO4·7H2O, 0.01% (w/v) FeCl3·6H2O, 0.005% (w/v) bromothymol blue, 0.005% (w/v) 0.005% (w/v) thymol blue, 1.5% (w/v) agar, autoclave at 110   °C for 20   min before adding 10   ml ampicillin (3   mg   ml−1), 10   ml sodium deoxycholate (10   mgml−1), 1% (v/v) ethanol; pH 8.6. Aeromonas develop as yellow colonies after incubation at 37   °C for 24   h.

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Rimler Shotts medium (RS): 0.05% (w/v) l-lysine hydrochloride, 0.65% (w/v) l-ornithine hydrochloride, 0.35% (w/v) maltose, 0.68% (w/v) sodium thiosulfate, 0.03% (w/v) l-cysteine hydrochloride, 0.003% (w/v) bromthymol blue, 0.08% (w/v) ferric ammonium citrate, 0.1% (w/v) sodium deoxycholate, 0.0005% (w/v) novobiocin, 0.3% (w/v) yeast extract, 0.5% (w/v) sodium chloride, 1.35% (w/v) agar; pH 7.0: After boiling to dissolve the ingredients, autoclaving is not required. Aeromonas develop as yellow colonies after incubation of spread plates of RS at 30   °C for 24   h.

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Rippey–Cabelli agar (mA): 0.1% (w/v) ferric chloride hexahydrate, 0.02% (w/v) magnesium sulfate heptahydrate, 0.2% (w/v) potassium chloride, 0.3% (w/v) sodium chloride, 0.5% (w/v) trehalose, 0.5% (w/v) tryptose, 0.2% (w/v) yeast extract, 1.5% (w/v) agar, 0.004%(w/v) bromthymol blue, 1% (v/v) ethanol, 20   mg   l−1 ampicillin, 100   mg   l−1 sodium deoxycholate, pH 8.0. Aeromonas spp. develop as yellow, circular, convex colonies.

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Starch–ampicillin agar (SAA): 0.1% (w/v) beef extract, 1% (w/v) proteose peptone no. 3, 0.5% (w/v) sodium chloride, 0.1% (w/v) starch, 1.5% (w/v) agar, 25   mg   l−1 of phenol red, 10   mg   l−1 of ampicillin. Putative Aeromonas colonies are 3–5   mm in diameter, and are yellow to honey pigmented. After flooding the plates with full or half strength Lugol's iodine, Aeromonas colonies will be surrounded by a clear zone, indicating such hydrolysis.

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Tryptone–soya–ampicillin broth (TSAB): tryptone soya broth containing 30   mg   l−1 ampicillin.

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Xylose–deoxycholate–citrate agar (XDCA): 1.25% nutrient broth no. 2, 0.5% (w/v) sodium citrate, 0.5% (w/v) sodium thiosulfate, 0.1% (w/v) ferric ammonium citrate (brown), 0.25% (w/v) sodium deoxycholate, 1.2% (w/v) agar, 1% (w/v) xylose, 0.0025% (w/v) neutral red; pH 7.0; dissolve by heating; autoclaving is not required. Aeromonas develop as colorless colonies.

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New approaches in microbial pathogen detection

L.N. Kahyaoglu , J. Irudayaraj , in Advances in Microbial Food Safety, 2013

Reverse transcription PCR (RT-PCR)

Reverse transcription PCR (RT-PCR), a modified form of PCR that allows the amplification of viral RNA, is currently the most sensitive and widely used method for foodborne virus detection (Casas and Sunen, 2001; Morales-Rayas et al., 2010). However, the application of this technique for routine analysis of food matrices is elaborate due to the need for sample concentration and the presence of residual food-related PCR inhibitors (Sair et al., 2002). Since only low numbers of viruses are present in food, inhibition is a more serious issue (Morales-Rayas et al., 2010). Therefore, several methods have been developed to concentrate and purify viruses and remove inhibitors from food samples before RT-PCR (Dubois et al., 2002; Croci et al., 2008).

The sample preparation procedures for detecting viruses in food typically involve one or more of the following: (i) elution of the virus particles from the food using a variety of buffers and solutions including solutions of glycine and sodium chloride, borate and beef extract, saline and beef extract, and beef extract alone; (ii) extraction with an organic solvent, most commonly with Freon to remove insoluble or poorly soluble organic compounds in the water; (iii) concentration of the viruses using sedimentation by antibody or ligand capture, flocculation, ultra-centrifugation or precipitation (commonly polyethylene glycol precipitation); and (iv) extraction of viral nucleic acids (there are two main approaches using phenol: chloroform extraction and guanidinium isothiocyanate extraction) ( Cook and Rzezutka, 2006; Goyal, 2006; Rodriguez-Lazaro et al., 2007). Various strategies have been proposed to improve the performance of each step over the years.

There are several commercial kits for nucleic acid purification, which are reliable, produce reproducible results and are easy to use. Most of these kits are based on guanidinium lysis and the capture of nucleic acids on a column or bead of silica (Bosch et al., 2011). However, sample preparation methods still require improvement to isolate viral particles from diverse food matrices without decreasing the sensitivity of the molecular method used for detection (Morales-Rayas et al., 2010).

The sensitivity and specificity of RT-PCR assays depends mainly on primer selection (Atmar and Estes, 2001). The major obstacle in NV detection with PCR arises from the very high genomic diversity of NV since new variants continue to evolve constantly (Widen et al., 2011). Therefore, it is difficult to select a single or even a small number of probes that can detect all possible NV variants (Atmar and Estes, 2001). Although ORF1 of the RdRp gene has been targeted in most of the assays (Nakayama et al., 1996; Jiang et al., 1999), the ORF1-ORF2 region has also been shown to be well conserved and is used in several assays (Katayama et al., 2002; Hohne and Schreier, 2004; Jothikumar et al., 2005b). One of the first enteric viruses detected by RT-PCR was HAV (Jansen et al., 1990). The VP1 capsid region was previously commonly targeted by primers in HAV detection; however, nowadays the 5' non-coding region is highly preferred for targeting. It has similar performance as VP1, approximately 1 RNA copy per reaction (Sanchez et al., 2007). For HEV detection, various specific sets of primers have been developed to amplify conserved regions within ORF1, ORF2 and ORF3 (Enouf et al., 2006). Most of the RT-PCR assays developed for rotaviruses target the structural genes VP4, VP6 and VP7 (Atmar, 2006). The hexon gene in adenoviruses is most commonly used as the target in PCR assays; it has been shown to be reactive in all adenovirus species (Jothikumar et al., 2005a; Atmar, 2006). More recently, a FRET-based real-time assay, which amplifies the adenovirus fiber gene, was described. It showed slightly better performance in terms of detection limits of AdV40 and AdV41 compared to TaqMan assays (Jothikumar et al., 2005a).

The major limitation of RT-PCR is its inability to distinguish between infectious and non-i nfectious viruses (Richards, 1999). Integrated cell culture PCR (ICC-PCR) and ICC/strand-specific RT-PCR have been proposed to compensate for this problem (Atmar, 2006; Jiang et al., 2004). ICC/strand-specific RT-PCR is a combination of cell culture and molecular biology-based methods, which requires initial propagation of infectious virus particles in a cell culture and the detection of a negative-strand RNA replicative intermediate as an indicator of viral replication (Jiang et al., 2004). The limitations of RT-PCR were eliminated in environmental samples by increasing the equivalent sample volume and thereby reducing the effects of inhibitory compounds (Reynolds et al., 1996). ICC-PCR and ICC/strand-specific RT-PCR assays targeting the VP3 genes, which code for a major HAV capsid protein, have been developed to detect viruses in water (Jiang et al., 2004). The ICC/strand-specific RT-PCR used in this study was demonstrated to be a novel, rapid, sensitive and reliable method, since it can detect infectious HAVs at inoculation level of 100 TCID50 per flask within four days in water samples.

Even though RT-PCR is a rapid and sensitive method and can detect viruses that are difficult or impossible to culture (Casas and Sunen, 2001), several different types of RT-PCR have been developed to improve the specificity and sensitivity of the standard method for foodborne virus detection such as nested RT-PCR (Love et al., 2008; Croci et al., 1999) and multiplex RT-PCR (Rosenfield and Jaykus, 1999; Formiga-Cruz et al., 2005; Coelho et al., 2003).

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Bioactive Natural Products

Tomáš Řezanka , ... Jan Masák , in Studies in Natural Products Chemistry, 2012

Animals

Animal kingdom does not belong to the priority groups synthesizing natural products. Still, compounds are described that have the ability to influence microbial QS.

Solenopsin A (96), a venom alkaloid from the fire ant Solenopsis invicta, efficiently disrupted P. aeruginosa QS signaling [144]. Biofilm formation in P. aeruginosa was gradually reduced in the presence of solenopsin A in a dose-dependent manner.

Ground beef extract contains several fatty acids such as palmitic acid ( 97), stearic acid (98), oleic acid (99), and linoleic acid (100) that were able to inhibit AI-2 activity. A mixture of these fatty acids, prepared at concentrations equivalent to those present in the ground beef extract, negatively influenced E. coli K-12 biofilm formation. Similarly, fatty acids isolated from poultry meat added in the appropriate ratio had concentration-dependent effect on the AI-2 inhibition of V. harvey BB 170 [145].

Bile acids (cholic acid (101), etc.) promote V. cholerae biofilm formation. The reason is probably an effort to reduce bacteria toxicity of this substance, which is quite significant against planktonic cells. Bile acid induction of biofilms was found to be dependent on the vps genes, which are responsible for the synthesis of EPS [146].

S. aureus is a versatile human and animal pathogen, commonly associated with catheter-related bloodstream infections. Biofilm formation by several S. aureus strains is stimulated by heparin (102), highly sulfated glycosaminoglycan. The exact mechanism of its effect on S. aureus adhesion is unknown [147].

Traditional Chinese medicine extensively uses extracts from Nidus Vespae (the honeycomb of Polistes olivaceous, P. japonicus, and others). The chloroform/methanol fraction of these materials showed the highest antibiofilm activities against S. mutans, the principal etiological agent of dental caries [148].

Application of honey in traditional medicine is also widely known. Alnaqdy et al. [149] studied its effect on adhesion of Salmonella interitidis to isolated intestinal epithelial cells in vitro. Bacteria pretreated with dilute solutions of honey up to a ratio of 1:8 had a significantly reduced ability of adhesion to epithelial cells. Chestnut honey and especially its aqueous extract have the ability to both degrade AHLs and inhibit the AHLs production of a number of bacterial strains. The result of such exposure was a significant suppression of biofilm formation by Erwinia carotovora, Yersinia enterocolitica, and Aeromonas hydrophyla [150].

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Flavour development in meat

J.S. Elmore , D.S. Mottram , in Improving the Sensory and Nutritional Quality of Fresh Meat, 2009

5.2.4 Character impact compounds

Not all of the volatile compounds found in meat contribute to its flavour and aroma. The character impact compounds are those compounds which make an important contribution to defining the typical aromas of cooked meat (Gasser and Grosch, 1988). Much of the work in this area has used the technique of aroma extract dilution analysis (AEDA), which will be described later in the chapter.

Beef was the first meat studied using this technique. Seventeen compounds in the cooked beef had high aroma values, of which 2-methyl-3-furanthiol and bis(2-methyl-3-furyl) disulphide had meat-like aromas. Both of these compounds are found in commercial meat flavourings (Ruther and Baltes, 1994). (E)-2-Nonenal and (E,E)-2,4-decadienal, which are formed from lipid breakdown, were the main contributors of fatty aromas to the cooked beef (Gasser and Grosch, 1988).

When an extract of stewed beef juice was analysed by AEDA, 4-hydroxy-2,5-dimethyl-3(2H)-furanone (furaneol), methanethiol and 12-methyltridecanal were found to be character impact components (Guth and Grosch, 1994 ). When the 15 compounds with the highest aroma values in the extract were dissolved in coconut oil, the aroma of the resulting sample bore a strong resemblance to stewed beef juice. Stewed beef was analysed by a vacuum distillation technique, whereas the cooked beef extract described above was analysed by simultaneous distillation/extraction. As the latter technique is unsuitable for the analysis of highly volatile compounds such as methanethiol, and polar compounds such as furaneol, it is likely that the compounds identified as stewed beef juice character impact compounds would be more representative than those identified in the cooked beef experiment.

3,5-Dimethyl-2-ethylpyrazine was reported as being an important contributor to both roast beef (Cerny and Grosch, 1992) and shallow-fried beef (Specht and Baltes, 1994). Other compounds contributing to the aroma of both types of meat included the lipid-derived compounds γ-octalactone, (E)-2-nonenal, (E,E)-2,4-decadienal and 1-octen-3-one, and the Maillard-derived compounds 3-(methylthio) propanal (methional) and 2,3-diethyl-5-methylpyrazine. Furaneol, 2-methoxyphenol (guaiacol) and 2-acetyl-2-thiazoline were all important contributors to roast beef, while 3,5(or 6)-dimethyl-2-vinylpyrazine and 2,5-dimethyl-3-ethylpyrazine were important in fried beef.

Less work has been carried out on the character impact compounds of pork, lamb and chicken. When cooked chicken was compared with cooked beef, 2-methyl-3-furanthiol, 2-furanmethanethiol (furfuryl mercaptan) and methional were important compounds in both meats. Bis(2-methyl-3-furyl) disulphide was much more important in beef aroma, while 2,5-dimethyl-3-furanthiol, another meaty aroma compound, and 2,4,5-trimethylthiazole, an earthy-smelling compound, were much more important in chicken aroma. (E,E)-2,4-Decadienal, γ-dodecalactone and nonanal were lipid-derived compounds which were also important in cooked chicken aroma (Gasser and Grosch, 1990). Furfuryl mercaptan, methional and (E,E)-2,4-decadienal were important in boiled chicken, along with furaneol, 4,5-dimethyl-3-hydroxy-2(5H)-furanone (sotolon), acetic acid, butyric acid and 2-acetyl-2-thiazoline (Kerler and Grosch, 1997).

Methanethiol has been shown to be important in cooked beef, pork and chicken, while 2-methyl-3-furanthiol and furfuryl mercaptan were both important contributors to cooked beef and pork aroma (Kerscher and Grosch, 2000). Rota and Schieberle (2006) showed that furaneol was a key contributor to cooked mutton aroma but did not contribute to raw mutton aroma, whereas 4-ethyloctanoic acid and trans-4,5-epoxy-(E)-2-decenal were important in both raw and cooked mutton aroma. When comparing beef, pork and chicken, Kerscher and Grosch (2000) stated that odour differences among the three species were mainly due to different concentrations of the key odorants; only a few compounds, e.g., 12-methyltridecanal and dimethyl sulphide in beef and hydrogen sulphide in chicken, have an impact on the aroma of only one species. Figure 5.7 gives the structures of those compounds described above which have not been present in any of the previous figures.

Fig. 5.7. Some character impact compounds of cooked meat.

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Developments in improving the safety of sprouts

S. Morabito , in Advances in Microbial Food Safety, 2015

14.2 Trends in the consumption of raw vegetables

In the last century, the habit of eating raw vegetables, including fruits, nuts and seeds, as well as raw fish, meat and milk has increased, and in some extreme cases, the entire diet is based on the consumption of such commodities.

During the nineteenth century, in the wake of the discoveries on the protein structure and the development of efficient methods to produce beef extract from carcasses, the consumption of meat increased exponentially, mainly among the new urban bourgeoisie, coming to be the epitome of a healthy diet. At the opposite extreme, the urban and rural poor population only had access to a diet composed essentially of white bread, sugar, gin or beer, with no meat. As a reaction to such an asymmetrical access to food resources a counter-movement, concerned for social justice, emerged. Initiated in Germany during the 1820s and known as ' lebensreform', such a movement became in the last decade of the 19th century a real mass movement pointing towards a 'moral physiology' based on natural health, temperance and vegetarianism. In this context, the Swiss practitioner Max Bircher-Benner, the inventor of muesli cereal, pioneered nutritional research and shaped a therapeutic programme based on the assumptions that anything eaten raw had a higher energy and nutritive value than anything cooked (Meyer-Renschhausen and Wirz, 1999) and that vegetables were to be the basis of this diet, because they were able to directly transform the energy of the sunlight into carbohydrates (Bircher-Benner, 1930). Bircher-Benner's theories were not welcomed when he introduced them to the medical establishment in 1900 but, in spite of the disgust raised in the medical community, his ideas found a fertile ground for their diffusion amongst those who were convinced that such a nutritional approach would rescue them from the meat-based culture. His theories, therefore, laid the foundations for the current dietary discipline known as raw-foodism.

Nowadays, the World Wide Web has increased the availability of information and web-based social media now constitute the most browsed sources of information for ordinary people concerned about public health issues affecting residents in high-income countries, such as obesity, cardiovascular diseases and cancer. The ease of access and dissemination of such information has allowed the development, on a global scale, of the social viewpoint that these concerns need alternative solutions to those offered by the medical and pharmacological establishment and that those solutions can be found through healthy nutrition. Today, the quest for healthcare answers by complementary and alternative approaches is a transcontinental experience, encompassing a wide range of people from those who are simply curious to those who perceive the traditional medical treatments as ineffective or even detrimental to human health.

To assess the legitimacy of such standpoints is out of the scope of this chapter. Nevertheless, it is undeniable that a raw food industry has grown based on the use of sprouts in response to an increasing consumer' demand for food commodities more in keeping with a holistic vision of life. Sprouts are celebrated as an important source of energy and vitamins, representing the essence of a healthy lifestyle and they are central in almost all the raw food-based diets such as the raw vegetarianism or the more radical dietary regimen known as raw veganism, up to the extreme 'sproutarianism', which is based almost entirely on sprouted foods (http://www.thesproutarian.com/).

In the European Union (EU), the commercial sector for sprouts has an appraised market value around 500   M€ per year at the consumer level with about 100 producers in the whole EU (EFSA, 2011a). However, such figures are approximate and most probably are underestimated. A precise assessment cannot be achieved owing to the existence of a high number of small producers, which are not surveyed and the habit, widespread in some EU countries, of growing sprouts at home. For sprouts consumption, data are difficult to collect, mainly because of the lack of awareness by consumers of when they are eating such a commodity. Sprouts are frequently consumed within composite preparations without being the main ingredient or used as garnishings to give dishes a better appeal. The data on sprouts consumption in the EU are gathered in the EFSA comprehensive European food consumption database (EFSA, 2011c). From the analysis of the information collated in such a repository, large variations in the figures related to the different Member States are observed, both in the percentage of subjects declaring that they consume sprouts and in the average daily amount of product consumed. Such differences may account for the diverse proportions of people in the habit of eating sprouts in different European countries or they may be related to the already mentioned lack of awareness about the presence of this product in mixed salads or other meals. It is not easy to identify sprouts in a composite meal. Such a commodity exists in different forms produced from a variety of different seeds species including, alfalfa, broccoli, adzuki beans, fenugreek, buckwheat, cabbage, clover, leek, lentils, linseed, mung beans, mustard, garlic, quinoa, radish, red beet, rice, rye, sesame, snow pea, soy, sunflower, triticale and wheat, grass pea, green and yellow peas, and onion (Beales, 2004, Schrader, 2002). Apart from the seed species another point of confusion is represented by the stage of maturity the plants are left to grow to. At least three different sprouted seeds commodities are recognized, namely sprouts, shoot and cress. The difference between sprouts and shoot is in the stage of growth, with the former being harvested just after germination and the latter grown to the stage of young plant. Both sprouts and shoots are developed without any substrate and using just water. Cress is grown to the same stage as shoots but on a solid support such as soil or other synthetic substrates.

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Sublethal injury, pathogen virulence and adaptation

D. Nyachuba , C. Donnelly , in Understanding Pathogen Behaviour, 2005

7.3.6 Bacillus cereus

Bacillus cereus is a common food contaminant. Effective control measures depend on destruction by a heat process and temperature control to prevent spore germination and multiplication of vegetative cells in cooked, ready-to-eat foods.

Faille et al. (1997) studied the effect of supplementing the recovery medium with lysozyme, glucose, NaCl and MgSO4 on the apparent heat resistance of four strains of B. cereus. The composition of an optimal medium (allowing the germination and growth of all surviving spores) and of a selective medium (inhibiting injured spores) was then determined: the optimal medium consisted of nutrient agar supplemented with 50 × 10−6 g lysozyme and 0.5 g MgSO4 per liter. The selective medium had the same composition but was supplemented with 15 g NaCl per liter. The injury and lethality of heat treatment of spores were analyzed for four B. cereus strains in both phosphate buffer and mechanically separated poultry meat. The four strains tested exhibited significant differences in injury and death rates, and considerable differences could be observed in survivor counts when spores were suspended in buffer or in meat. Moreover, variability in behavior when the strains were heat treated in buffer could not be extrapolated to the same strains suspended in food. These findings indicate that it is necessary to be very careful when evaluating potential hazards from B. cereus spores in various foods. To evaluate the efficiency of heat treatments, heating medium and strain types must be considered.

B. cereus spore stress survival and impact of stresses on recovery

Johnson and Busta (1983) examined B. cereus survival during heating and cooling. They found that spore response could not always be predicted with data generated at constant temperature. After a rapid heating (900 °C/h) to 90 °C, spores were inactivated in two distinct temperature ranges during cooling at rates of 5 or 10 °C/h. Thermal inactivation occurred during cooling from 90 to 80 °C; the population remained stable during cooling from 80 to 50 °C; and a second period of inactivation occurred during cooling from 50 to 35 °C. However, when spores were heated more slowly (20 or 40 °C/h), inactivation that occurred at the lower temperatures was not observed. This phenomenon was observed with three of four B. cereus foodborne illness-related strains studied. The apparent low temperature inactivation of spores observed during cooling from 90 °C has not been elucidated. This inactivation is believed to represent injury rather than death of spores. If this is true, potentially viable spores may remain undetected with standard microbiological analysis. Repair of these injured spores in food and subsequent growth may lead to potentially hazardous situations (Busta, 1976).

Heating induces a number of changes in spores through the process of activation (Berg and Sandine, 1970): increased metabolic activity, faster germination rates, less exacting germination requirements and changes in spore proteins and enzymes (Johnson and Busta, 1984). During germination, spores lose heat resistance and refractility.

Johnson and Busta (1984) showed that initial heat treatments can induce subsequent temperature sensitivity in B. cereus spores during outgrowth. Attempts to repair damage and speculation on the metabolic function involved in heat-induced temperature sensitivity was made. Inactivation of B. cereus spores during cooling (10 °C/h) from 90 °C occurred in two phases. One phase occurred during cooling from 90 to 80 °C; the second occurred during cooling from 46 to 38 °C. In contrast, no inactivation occurred when spores were cooled from a maximum temperature of 80 °C. Inactivation of spores at a constant temperature of 45 °C was induced by initial heat treatments from 80 to 90 °C. The higher temperatures accelerated the rate of inactivation. Germination of spores was required for 45 °C inactivation to occur. However, faster germination was not the cause of accelerated inactivation of spores receiving higher initial heat treatments. Repair of possible injury was not observed in TSB (BBL Microbiology Systems), peptone, beef extract, starch or l-alanine at 30 or 35 °C. Microscopic evaluation of spores outgrowing at 45 °C revealed that when inactivation occurred, outgrowth halted at the swelling stage. Inhibition of protein synthesis by chloramphenicol at the optimum temperature also stopped outgrowth at swelling, suggesting that disruption of protein synthesis may be the reason behind the 45 °C inactivation observed.

Gonzalez et al. (1997) evaluated the effects of the addition of starch, glucose, sodium chloride (NaCl), sodium citrate (Na3C6H5O7•2H2O), monopotassium phosphate (KH2PO4) and disodium phosphate (Na2HPO4•12H2O) to the recovery medium on apparent heat resistance of B. cereus spores (ATCC 4342, 7004 and 9818). They found that Na3C6H5O7•2H2O, Na2HPO4•12H2O at concentrations of 0.1 % were effective inhibitory agents for heat injured B. cereus spores, especially for strain 9818, although only KH2PO4 and Na2HPO4•12H2O caused a significant reduction (P < 0.05) in D-values obtained for strain 9818. NaCl also had a marked effect on the recovery of heat-injured spores, with levels as low as 0.5 % resulting in a significant reduction in recovery rates for strains 9818 and 7004. In all cases, increasing the NaCl levels from 0.5 to 4 % resulted in a progressive decrease in spore recovery. D-values gradually decreased as the salt content increased, although the concentrations that produced statistically significant differences (P < 0.05) varied among strains. The addition of starch at 0.1 % resulted in a significant increase in the counts for strains 9818 and 7004. In contrast, glucose (0.1 %) did not significantly affect the counts obtained. Neither of these compounds affected decimal reduction times. Also, no statistically significant (P > 0.05) differences were detected among z-values for the spores of the three strains recovered in the presence of different additives assayed. z-Values ranged from 6.67 to 8.32, with a mean value of 7.56 ± 0.46 °C.

Gonzalez et al. (1996) have also investigated the influence of pH of the recovery medium, in the range 7.6–5.4, on the apparent heat resistance of three strains of B. cereus (ATCC 4342, 7004 and 9818). The highest counts of heat-injured spores were obtained at pH near neutral (pH 7.0). Counts decreased markedly as pH was reduced, especially with longer heating times. When the media were acidified, the apparent D-values tended to decrease, although some exceptions related to the strain and the nature of the medium were observed. The pH of the medium did not affect z-values determined during this study.

In another study by Gonzalez et al. (1995), the effects of recovery media and incubation temperature on the apparent heat resistance of three ATCC strains (4342, 7004 and 9818) of B. cereus spores were examined. Nutrient agar (NA), TSA, plate count agar (PCA) and milk agar (MA) as the media and temperatures in the range of 15–40 °C were used to recover heated spores. Higher counts of heat-injured spores were obtained on PCA and NA. The optimum subculture temperature was about 5 °C below the optimum temperature for unheated spores. There were no significant differences in heat resistance with the different recovery conditions except for strains 4342 and 9818 when MA was used as the plating medium. Large differences in D-values were found among the strains (100D = 0.28 min for 7004; 100D = 0.99 min for 4342; 100D = 4.57 min for 9818). The 7004 strain showed a subpopulation with a greater heat resistance. The z-values obtained for the three strains studied under the different recovery conditions were similar (7.64°C ± 0.25).

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Biotechnological Production of Enzymes Using Agro-Industrial Wastes

N. Gopalan , K.M. Nampoothiri , in Agro-Industrial Wastes as Feedstock for Enzyme Production, 2016

Upstream Processes

The primary raw material used for fermentative production of enzymes can come from any source of agricultural by-products obtained from cereal crops, spice crops, seasonal crops, oil seeds, fruits, vegetables, roots and tubers, postharvested plant materials and processing industries, etc. Traditional bioprocesses utilized refined sugars as carbon sources and salts containing nitrate or ammonium groups as nitrogen source, with certain processes using undefined nitrogen sources, such as yeast extract and peptone and beef extract. The ratio of the carbon to the nitrogen source plays a significant role in the yield of enzymes or any bioproduct, along with other factors present in the medium ( Aiyer, 2005). Later, with the focus being shifted to make different bioprocesses economical, cheaper sources of carbon, such as cane molasses and lignocellulosic substrates or starch/starch hydrolysates, came to light (Furlan et al., 2000; Olsson et al., 2003). One such example is, cellulase production by Aspergillus fumigatus grown on mixed substrate of rice straw and wheat bran (Sherief et al., 2010). Slowly, bioprocesses using cheaper nitrogen sources, like corn steep liquor and urea, started to take center stage (Edwinoliver et al., 2009; Nascimento et al., 2009).

Agricultural/agro-industrial wastes may be rich in carbohydrates, proteins, or lipids and it may are available in solid or liquid forms. Any agricultural waste if used as a major component of the fermentation medium can reduce the total cost of production by decreasing the cost of the raw material used for the fermentation process. In biomass-based technology, costs associated with upstream processing of enzyme production involve the cost of the raw material, if there is any pretreatment (such as chemical or enzymatic) of raw material, transport of the raw material to production site, and associated labor and energy costs, etc. The nature of the raw material like starchy material or cellulosic material itself affects the fermentation/production process as a whole. Choosing appropriate, readily available, and cheaper substrates for the desired enzyme is a must. Preferably, the raw material could be a good inducer for the enzyme production as well. The best example is that of lignocellulosic materials serving as substrate and inducer for the production of cellulase enzymes (Lau et al., 2012). Raw material costs for a typical bioprocess range from 10% to 50% of the TCP, and hence using agro-residual wastes would be beneficial, as more capita can be allotted to other components of the process and recurring costs on raw materials could be reduced (Castilho et al., 2000).

Similarly, depending on the mode of fermentation, viz., solid state fermentation (SSF) or submerged fermentation (SmF), the infrastructure, and consequent maintenance will vary. Equipment used in the pretreatment of raw materials involve sterilization or pasteurization methods to decrease or eliminate the competing microbes already present on/in the fermentation medium/nutrient medium. Depending on the mode of fermentation involved for the process, the equipment and the scales for this purpose may vary. Generally for SSF, small-volume, high-temperature, and pressure-based sterilization equipment is used, which is economical. For SmF, the equipment generally is coupled with the fermentation vessel in the form of a temperature-exchange jacket, whose price is inclusive in the cost of the fermenter itself. Raw materials require additional pretreatment steps in certain cases, viz., defatting or destarching when the microorganism is required to produce enzymes free of lipases and amylases, eg, feruloyl esterases, require destarched wheat bran as a substrate or defatted oil cakes (Bonnin et al., 2002; Laszlo et al., 2006), or wet heat pretreatment, in which case additional equipment or associated facilities may be required (Palmqvist et al., 1997; Singh et al., 2011).

Depending on the course followed, there are differences in the economic ramifications of the process. Submerged fermentation involves the cultivation and subsequent production of the enzymes produced by the microorganism while suspended in a nutrient-rich fluid medium. The energy requirements to maintain the temperature of large quantities of liquids are high, along with increases in the cost of sterilizing or other pretreatment required for the process. Submerged fermentation processes have been used classically for major fermentative processes since the onset of industrial microbiology, starting with the fermentative production of antibiotics from fungi and actinomycetes during the era of World War II. Fermentative production of some carbohydrases and lipolytic enzymes through SmF of agro-wastes have been reported (Sharma and Satyanarayana, 2006; Teng and Xu, 2008; Singh et al., 2009; Vidyalakshmi et al., 2009; Nagar et al., 2010). Various reactor designs are available for enzyme production processes. However, the stirred tank reactor is generally preferred over other designs. Stirred tank reactors are closed reactors that are expensive and the costs incurred for setting these normally takes the top spot in the TCP (Castilho et al., 2000). The stirred tank reactor also causes an increase in the costs incurred to efficiently handle bulk liquid quantities. Overall, the equipment costs for SmF are very high compared to SSF. Labor required for running stirred tank reactor-based bioprocesses is generally confined to trained personnel in the field of bioprocess engineering (Max et al., 2010). Since the specific heat of water, which makes up for most of the mass of the fermentation medium, is very high, the heating and cooling cost for the operation of SmF processes would rise. If the batch time for fermentation is small, smaller volume fermenters may be employed. However, for processes with longer incubation periods, the fermenter volume should be increased to compensate for productivity, concurrently leading to increased cost of equipment used. Increasing the number of batches/fermenters running parallel may be desirable when downstream processes have a smaller batch time compared to the fermenter batch time.

Solid-state fermentation is the preferred method for production of various enzymes from agro-industrial substrates, especially when the substrates are insoluble in water. Solid-state fermentation is defined as a process where a microbe is grown on a substrate in the absence or near absence of free water. SSF is preferred over SmF because it was revealed that cultures grown in solid state do not show catabolite repression (Viniegra-González et al., 2003; Viniegra-González and Favela-Torres, 2006) that is normally a prevalent phenomenon in SmF, and as a result enzyme concentrations that are usually unattainable in SmF are encountered in SSF. Sensitivity to ionic contaminants in water is reduced in case of solid-state cultivation of microbes, reducing the dependency of processed water for the bioprocess (Shankaranand and Lonsane, 1994). SSF is carried out on simple tray fermenter, made of wood or aluminum (Fig. 14.1). Tray fermenters are cheap alternatives compared to submerged fermenters. The process of SSF involves wetting of the solid substrate with nutrient media to a certain initial moisture level (50–90%) (Pandey, 2003). Since the microbe is grown on a solid substrate, normally the temperature and humidity is controlled for the whole unit where the SSF is carried out. Trays are essentially open fermenters, as lower moisture content and high amounts of inoculums ensures the absence of contamination. The labor involved with running an SSF does not require trained personnel; cheap labor force under the supervision of a moderately trained personnel suffices for smooth operation of batches. The costs incurred for cooling and other energy-based expenditures are lesser compared to SmF, as the specific heat capacity for water is very high and water makes up a very small percentage of the medium for SSF compared to SmF. SSF process is carried out with no control over the pH, while SmF processes require precise pH control using acids and alkalis. The process cannot be controlled as precisely as SmF, due to the heterogeneous nature of the fermentation medium, however, from the economic standpoint, since the process requires lesser control than SmF, and energy expenditure as well as lesser inputs for the raw material, SSF becomes a very viable option.

Figure 14.1. Filamentous fungi growing on agro-residual solid substrate (wheat bran)—a low-cost enzyme production strategy.

Upstream process requires the use of a robust microbial culture. Bacteria or fungi can be used for either SmF or SSF. Ideal industrial cultures are fairly immutable, produce either large amounts of enzyme, or produce in higher concentrations than relative wild-type varieties. When in relation with an enzyme as a product, the microbe involved varies with the final use of the enzyme. For example, amylase as an enzyme is used for hydrolyzing starchy substrates under benign conditions. However, different variants of the amylase enzyme produced by different organisms are used for different applications. Amylase used for saccharification of waste starchy biomass for further fermentation into ethanol uses ordinary amylase, while amylases used for baking may use amylase mixes that impart flavors to the bread, by virtue of other enzymes (mostly esterases) produced by the particular organism. Amylase treatment improves bread shelf life by modifying the starch structure (Martínez-Anaya, 1996). Therefore, procuring the right organism according to the target market is one of the most important investments that an enzyme producer has to carry out. Isolating an industrial strain from nature is not only a tedious process but also requires considerable capital investment. A procured microbe is a catalogued organism with most of the parameters known to the fermentation expert, hence guarantees tighter control over the process of fermentation. Table 14.1 lists some of the commercial strains for production of enzymes.

Table 14.1. List of Industrially Important Enzymes and Potent Producers

Enzyme Organisms
Aminopeptidases Clostridium histolyticum, Vibrio proteolyticus, Aspergillus
Alpha-amylase Bacillus amyloliquefaciens subsp. amyloliquefaciens
Amylase Aspergillus foetidus, B. amyloliquefaciens subsp. amyloliquefaciens, Bacillus licheniformis, Endomyces fibuliger, Paenibacillus macerans, Paenibacillus polymyxa, Rhizomucor miehei, Rhizomucor pusillus, Thermoanaerobacter brockii subsp. finnii, Thermoanaerobacter ethanolicus
Amylase, alkaline Bacillus halodurans, Bacillus pseudofirmus, Bacillus sp.
β-amylase, alkaline Bacillus halodurans
Amylase, halophilic Nesterenkonia halobia, Amylase, thermoacidophilic, Alicyclobacillus acidocaldarius subsp. acidocaldarius
Amylase, thermostable Bacillus sp., Geobacillus stearothermophilus, Thermoanaerobacterium thermosaccharolyticum, Thermoanaerobacterium thermosulfurigenes
l-asparaginase Cupriavidus necator, Escherichia coli, Wolinella succinogenes
Cellulase Cellulomonas uda, Chaetomium globosum, Clostridium alkalicellulosi, Clostridium thermocellum, Phanerochaete chrysosporium, Thermoascus aurantiacus, Trichoderma reesei, Aspergillus niger, Aspergillus flavus, Aspergillus terreus
Cellulase, alkaline Bacillus cellulosilyticus, Bacillus wakoensis
Cellulase, enhanced Trichoderma reesei, Bacillus sp. Escherichia coli, Kluyveromyces marxianus, Sulfolobus solfataricus
Invertase Clostridium pasteurianum, Rhizopus oryzae
Laccase Heterobasidion annosum, Hypholoma fasciculare, Pleurotus cystidiosus, Pleurotus ostreatus, Spongipellis litschaueri, Trametes versicolor
Lactase Kluyveromyces marxianus
Nitrilase Pseudomonas brassicacearum subsp. brassicacearum, Pseudomonas putida, Variovorax sp.
Pectinase Fusarium oxysporum, Pichia Canadensis, R. oryzae, Trichoderma reesei
Peroxidase Geotrichum candidum, Inonotus weirii, Phanerochaete chrysosporium
Protease Acremonium chrysogenum, Aneurinibacillus migulanus, Bacillus cereus, Bacillus circulans, Candida tropicalis, Coprinus cinereus, Coprinus radians, Dictyostelium discoideum, Lysobacter enzymogenes subsp. enzymogenes, Ruminobacter amylophilus, Streptomyces sp.
Protease, acid Candida parapsilosis, Candida tropicalis
Protease, alkaline Bacillus alcalophilus, Bacillus clausii, Bacillus licheniformis, Bacillus subtilis
Xylanase Phanerochaete chrysosporium

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