microbiological tests in spice industry

MICROBIOLOGY

Microbial infections of concern with respect to spices:

1. Bacterial infections of concern with respect to spices.

v Highest health hazard

• Salmonella typhi

• E.coli (pathogenic)

v Moderate health hazard

•Clostridium botulinum

• Bacillus cereus

• Other salmonella

2. Microbial intoxications

v Bacterial

• Bacillus cereus

• Staphylococcus aureus

• Clostridium botulinum

v Fungal

• Mycotoxins namely aflatoxins

Determination:

• Aerobic mesophilic bacterial count

• Yeast and mold count

• Indicators bacterial count the coliforms.

General procedures for microbial quality assessment:

ENUMERATION:

•Preparation of food homogenate

•Preparation of dilution

•Dispensing of required inoculums into

Petri plates

•Mixing with appropriate agar medium for

preparation of the gel.

•Incubation of plates

•Counting of colonies and interpretation

DETECTION:

•Enrichment with specific growth

Medium

•Plating on selective agar medium

•Detection of particular bacterial colony

on plates

•Biochemical conformation

•Serological conformation

VIBRIO CHOLARAE

MEDIUM:

1) Alkaline peptone water

2) TCBS agar

PROCEDURE:

Prepare 225ml alkaline peptone water, 25g sample is added to it. The solution is incubating 6 hours at 37°C. The prepared solution is loop to TCBS agar in petri plate and incubates 24 hours at 37°C. After incubation observe the growth of yellow colony.

SALMONELLA

DETECTION & IDENTIFICATION

INTRODUCTION:

Salmonella are gram negative nonsporing, facultative anaerobes that belong to the family enterobacteria. They are usually motive and produce both acid and gas from glucose. This rod- shaped bacteria is of important because it can cause food borne salmonellas (food poisoning) over 2,300 servers have been indentified and are distinguished according to their antigenic stricture. A wide variety of foods have been involved in food borne salmonellas, however, egg, poultry , meat and products have been the main vehicles.

SCOPE:

This method is applicable to processed and unprocessed foods and environmental samples. Only the pre-enrichment protocols ground and whole spices and herbs fruits, vegetables, seasoning blends, cheese, cheese bread, flour, flour based blends, onion, garlic & liquid whole eggs have been outlined. Salmonella is pathogen. It is imperative that standard laboratory safely practices be followed. Handle all inoculated media as bio-hazard materials

MEDIUM:

TSB

RV

TTB

XLD

BSA

HEA

TSI

LIA

SALMONELLA CONVENTIONAL

Pre enrichment (225ml/RSB, 25g

sample kept room temperature

for the (60+/5) 18-24 hr incubation)

			RV (0.1 sample – 10 ml media) TTB (1 ml sample)

Enrichment

Selective media

TSI, LIA media

Glucose Lysine Bio chemical identification (IMVIC Test)

XLD:

Suspect colonies are pink or red with or without black center of completely black, same atypical colonies are yellow with or without black center and should be picked for confirmation.

BSA:

Suspect colonies are dark brown, gray or block usually with surrounding black 30 ml and metallic sheet with little or nor darkening of the surrounding medium, same a typical colonies are green with little or nor darkening of the surrounding medium should be picker for confirmation.

HEA:

Suspect colonies are blue green with or without black centers or completely black, same typical colonies are yellow with or without black centers and should be picked for confirmation.

BIOCHEMICAL IDENTIFICATION:

SUBJECT:

IMVIC Test

INTRODUCTION:

An important group of tests to distinguish between the coliform bacteria as well as other organisms in the family enterobacteriaceae is known by the acronym

IMVIC, these tests determine whether an organism has the ability to produce:

a) Indole from tryptophan (I)

b) Sufficient acid to reduce the medium ph to below 4.4, the point of the indicator methyl red (M)

c) Acetoin (acetyl methyl carbind-voges proskaner)

d) The ability to and utilize citrate (C)

These tests may be used for the bio chemical conformation of E. coli as well as acid in the identification if the rapid.

SCOPE:

This method is applicable to all microbiological laboratories.

VIDAS- SALMONELLA (SLM):

VIDAS Salmonella is an automated quantitative test for use on the VIDAS instruments, for the detection of salmonella in food and environmental specimens, using the ELRA technique (Enzyme Linked Fluorescent Assay).

PRINCIPLE:

VIDAS salmonella is an enzyme immune assay for use on the automated VIDAS system for the detection of salmonella antigens using the ELFA. This solid phase Receptacle (SPR) serves as the solid phase as well as the pipetting device the interior of the SPR is coated with anti-salmonella antibodies soaked and pre-dispensed in the sealed reagent strips.

TECRA-SALMONELLA:

225ml mpw + 25g sample

¯ 16-20 hours incubation

Loading to kit (wells)

VIDAS (Vital Immune Diagnostic Assay System)

225ml BPW + 25 g sample (18-24 hours at 36°)

¯

1ml 0.1ml

TTB RV media (6-8 hr incubation)

1 ml

¯

m broth (post enrichment) 42-2°C

incubation (18-24 hrs)

¯

1ml +1ml 1ml -15 boiling water

then cool

T-tube

(95+100°c) ¯

VIDAS ®loading 0.5 ml to vidas

¯

XLD, BSA to confirm

All of the assay steps are performed automatically by the instrument. The reaction media is cycled in and out of the SPR several times.

Part of the enrichment broth is dispensed into the reagent strip. The antigen present will bind to the anti-salmonella are during the washing antibodies coating the inferior of the SPR. Unbound components are eliminated during the washing steps. Antibodies conjugated with alkaline phosphate are cycled in and out of the SPR and will bind to any salmonella antigens which are themselves bound to the antibodies on the SPR. A final wash step removes unbound conjugate. During the final detection step, the substrate (4methyl-umbelliferone) the fluorescence of which is measured at 450nm.

At the end of the assay, results are automatically analyzed by the instruments which is calculated a test value for each sample. This value is then compared to internal reference and each result is interpreted (positive, negative).

TUBE MOST PROBABLE NUMBER (MPN) COLIFORM,

FECAL COLIFORM, AND E. COLI DETERMINATION:

INTRODUCTION:

In general, coli form bacteria are represented by 4 genera of the family Enterobacteriaceae, Escherichia, Enterobacter.

They are gram negative, rod-shaped micro organisms that are commonly found in nature and the intestinal tract of animal humans. They have the capacity to ferment lactose with the production of gas and are able to grow in the presence of bile salts. Because of the genera case with which coli forms can be cultivated and differentiated they are used as indicator organisms to assess food safety and sanitation.

The modified to grow and on the ability of fecal coli forms to grow and produce bas at 45-5c in a lactose broth media. Escherichia coli are the coliform of most concern because its presence in food indicted fecal contamination or unsanitary practices during or after production.

SCOPE:

This is the traditional method for enumerating total coli forms, fecal coli forms and E. coli in foods.

DETERMINATION OF E. coli:

MEDIUM:

§ LSB medium

§ EC Broth

§ EMB agar

§ Macon key agar

PROCRDURE:

MPN coli form, fecal coli form, and E. coli determination:

Prepare sample dilutions. Using a sterile 10ml pipette, transfer 10.0 ml of the 10^-1 dilution into three tubes of double strength. Lauryl sulfate broth. This is equivalent to a 10° dilution. Using a sterile 1 or 2.0 ml pipette and working from the highest dilution to the lowest, transfer 1ml of each dilution into each of three tubes of single strength Lauryl sulphate broth. By working from the separate one for each dilution must be employed. Incubate the tubes at 35°C for 48±2 hours. A tube is presumptively positive if there is effervescence when the tube is gently agitated. Confirm each presumptively positive lauryl sulfate tube by transferring a loop full of the broth into a tube of brilliant green bile broth and into a tube of EC broth. Incubate the Brilliat green Bile tubes at 35±1°c for 48±2 hours and incubate the EC broth tubes in a 45.5±-021° water bath for 48±-2 hours. Production of gas in the BGB tubes incubate, confirmed coli form bacteria. Record the number of positive tubes for each dilution.

Tubes evidencing gas production in EC broth are considered positive for fecal coli forms. Record the number of positive tubes for each dilution. To confirm the presence of E. coli in EC positive tubes, streak Levine EMB agar plates with a loop full of broth from each positive tube of EC broth incubate for 24±-2 hours at 35 ±-1°C

After incubation, examine the plates for typical E. coli: colonies: small, wine colored with or without a gram metallic sheath.

STANDARD PLATE COUNT DETERMINATION:

INTRODUCTION:

The standard plate count or total plate count enumerates viable mesophilic aerobes capable of growth on designated media. the method which is highly empirical, is based on the assumption organism. The standard plate count method or aerobic plate count is one of major application of the colony count method. These procedures are based on the assumption that each microbial cell in a sample will form a visible, separate colony when mixed with an agar or other solid medium and permitted to grow. The microorganism found in foods, often represent the number of population with many growth requirements, it can be possible that some organism may not be able to grow under conditions employed consequently, the counts are, at best, an estimate and should not be reported as absolute.

SCOPE:

This method is applicable to all foods standard plate count aerobic mesophilic

MEDIUM-TPC (total plate count agar):

Prepare the buffer solution (make up 34 g phosphate buffer in 1000ml water). Take 100ml to each bottle. This solution is mix well in ‘8’ shaped 25 times. Take clean and sterilized Petri dishes and 1ml mixed solution is poured into the sample. The sample is then mixed clock wise and anticlockwise and up and down manner. It is then stand for setting the solution. Then incubate at 37°C for 48 hour. After two days observe the growth. The PH range is between 7.0 ± 2.

YEAST AND MOLD DETERMINATION:

MEDIUM:

PDA (Potato dextrose agar)

INTRODUCTION:

Both yeast and mold cause varying degrees of deterioration and decomposition of foods. The ability of fungi to spoil many foods is due to part to their relatively versatile environment requirement.

PROCEDURE:

Prepare the buffer solution. Take 100ml to each bottle. This solution is mix well in ‘8’ shaped for 25 times. Take clean and sterilized Petri dishes and 1ml mixed solutions poured into the Petri dishes. Prepared PDA media poured into the sample. The sample is then mixed clockwise and anticlockwise and up and down manner. It is then stand for few minutes on laminar air flow bench for 3-5 days. After five days count the number of yeast and mold.

instrumental methods in spice industry

INSTRUMENTATION

1.SPECTROPHOTOMETER:

1. CURCUMIN (Colour value)

PROCEDURE:

Weigh 0.2 gram turmeric powder in a 250 ml. RB flask, Add 75 ml acetone. Cover the RBF with aluminium foil paper to prevent light absorption. Reflux for 1 hour in a mantle using water condenser at 10^oC. Then cotton filter to a 250 ml. dark standard flask and make up with acetone. Then bowl pipette 5 ml to 50 ml standard flask and make up using acetone.

The Curcumin content is detected by spectrophotometer it contains two quvettes, one for acetone and one for solution which is used to make up the solution. The wave length of Curcumin is 420 nm. Adjust to that wave length and make the reading to 0. Then find the value of solution.

CALCULATION:

Absorbance x dilution factor

Factor derived from Curcumin standard x wt. of sample taken

2. PIPERINE (Pepper)

PROCEDURE:

Take 0.5 gm. Sample in 250ml RBF. Add 70 ml SDA No.3A denatured alcohol. (i.e. 20: 1 - ethanol: methanol solution) the flask is covered with aluminium foil. Reflex for one hour at 20^oC allow cooling to room temperature and making upto 250 ml. Use extraordinary filter paper and 250 ml amber colour std. flask. From this solution pipette 2 ml and make upto 50 ml. It is filled in cuvette and kept in spectrophotometer and the reading is noted at the wavelength of 342 nm.

CACULATION:

Absorbance x dilution factor

Std. of Piperine x weight of sample

3. CAPSANTHIN (Colour value)

PROCEDURE:

Accurately weigh 1 gm of ground chilly sample into a 100 ml volumetric flask. Add 75 ml extra pure acetone into this flask. Keep this inside a dark cupboard for 16 hours or overnight to allow the particles to settle. Take 5 ml into 100 ml volumetric flask. Make up with acetone and transfer a portion of the extract using Millipore and pour into quvette and keep in spectrophotometer and measure the absorbance (A) at 460 nm with acetone.

CALCULATION:

Absorbance x 60.4 x 10

Weight of sample taken

2.CHROMATOGRAPHY

OPERATION:

The sample to be analyzed is introduced in small volume to the stream of mobile phase. The analyte's motion through the column is slowed by specific chemical or physical interactions with the stationary phase as it traverses the length of the column. The amount of retardation depends on the nature of the analyte, stationary phase and mobile phase composition. The time at which a specific analyte elutes (comes out of the end of the column) is called the retention time; the retention time under particular conditions is considered a reasonably unique identifying characteristic of a given analyte. The use of smaller particle size column packing (which creates higher backpressure) increases the linear velocity (speed) giving the components less time to diffuse within the column, leading to improved resolution in the resulting chromatogram. Common solvents used include any miscible combination of water or various organic liquids (the most common are methanol and acetonitrile). Water may contain buffers or salts to assist in the separation of the analyte components, or compounds such as trifluoroacetic acid which acts as an ion pairing agent.

1. AFLATOXIN (CHILLY)

INSTRUMENT CONFIGRATION:

· Shimadzu HPLC with following configuration

· Liquid chromatograph pump

· Fluorescent detector

· Rheodine injector

· c- 18 250 mm 170 Armstrong silica column

· CMB- 20 alite system controller

· Data processor- LC solution

INSTRUMENT CONDITION:

· Mobile phase flow rate: 1.2 ml/min

· Total run time : 20 min

· Temperature : 25⁰C

MOBILE PHASE:

· Ethanol: Water (45:55)

AIM:

To detect the presence of Aflatoxin in chilly

PROCEDURE:

Take 25 gm sample and 5 gm sample into the blending jar. Add 100 ml 80% methanol. This is blended for 80 sec, filtered through fluted filter paper. From this solution 100 ml passes through immuno affinity column. 20 ml water washing, elute with 1ml methanol. This is collected in cuvette (1 ml). This colour solution is injected in HPLC and a graph is obtained which shows the absence or presence of Aflatoxin.

2. CAPSAICIN (Pungency)

INSTRUMENT CONDITION:

· Mobile phase flow rate: 1 ml/min

· Total run time : 20 min

· Temperature : 25⁰C

MOBILE PHASE:

· Acetonitrile: Water: Acetic acid

AIM:

To find the percentage of capsaicin in chilly powders

PROCEDURE:

Take 12.5 gm of chilly powder into a RBF and add 100 ml alcohol. Reflux for 3 hours in multimantle. Cool at room temperature. Filter it using whattmann No.1 filter paper, and then using Millipore. It is a device which has two kinds of filters. Collect the sample and inject through the inject port system.

3. SUDAN DYE (Adulteration)

INSTRUMENT CONFIGRATION:

· Shimadzu HPLC with following configuration:

· Liquid chromatograph pump

· UV-Visible detector

· Rheodine injector

· SCL-10 A VP system controller

· C- 18 250 nm 100 Armstrong 5 micron silican column

· Data processor- LC solution

INSTRUMENT CONDITION:

· Mobile phase flow rate: 1 ml/min

· Total run time : 20 min

· Temperature : 25⁰C

MOBILE PHASE:

· Acetonitrile: Water (95:5)

· Water: Acetic acid (100:16.5)

AIM:

To detect the presence of Sudan dye on chilly

PROCEDURE:

Take 10 gm sample into 250 ml conical flask then add 100 ml CH₃CN. This solution is stirred for 1 hour using magnetic stirrer. Then filter through whattmann No.1 filter paper from this solution pipette 5 ml make up to 50 ml using acetonitrile. Then it Millipore filtered. Take this sample and inject into the HPLC. A graph is obtained which shows the absence or presence of Sudan dye.

GAS CHROMATOGRAPHY

Gas-liquid chromatography (GLC), or simply gas chromatography (GC), is a type of chromatography in which the mobile phase is a carrier gas, usually an inert gas such as helium or an unreactive gas such as nitrogen, and the stationary phase is a microscopic layer of liquid or polymer on an inert solid support, inside glass or metal tubing, called a column. The instrument used to perform gas chromatographic separations is called a gas chromatograph (also: aerograph, gas separator).

Gas Chromatography is different from other forms of chromatography (HPLC, TLC, etc.) because the solutions travel through the column in a gas state. The interactions of these gaseous analyte with the walls of the column (coated by different stationary phases) causes different compounds to elute at different times called retention time. The comparison of these retention times is the analytical power of GC. This makes it very similar to high performance liquid chromatography.

GC analysis

A gas chromatograph is a chemical analysis instrument for separating chemicals in a complex sample. A gas chromatograph uses a flow-through narrow tube known as the column, through which different chemical constituents of a sample pass in a gas stream (carrier gas, mobile phase) at different rates depending on their various chemical and physical properties and their interaction with a specific column filling, called the stationary phase. As the chemicals exit the end of the column, they are detected and identified electronically. The function of the stationary phase in the column is to separate different components, causing each one to exit the column at a different time (retention time). Other parameters that can be used to alter the order or time of retention are the carrier gas flow rate, and the temperature.

In a GC analysis, a known volume of gaseous or liquid analyte is injected into the "entrance" (head) of the column, usually using a micro syringe (or, solid phase micro extraction fibers, or a gas source switching system). As the carrier gas sweeps the analyte molecules through the column, this motion is inhibited by the adsorption of the analyte molecules either onto the column walls or onto packing materials in the column. The rate at which the molecules progress along the column depends on the strength of adsorption, which in turn depends on the type of molecule and on the stationary phase materials. Since each type of molecule has a different rate of progression, the various components of the analyte mixture are separated as they progress along the column and reach the end of the column at different times (retention time). A detector is used to monitor the outlet stream from the column; thus, the time at which each component reaches the outlet and the amount of that component can be determined. Generally, substances are identified (qualitatively) by the order in which they emerge (elute) from the column and by the retention time of the analyte in the column.