Saturday, November 15, 2014

Thesis: Pork Shelf Life Extension Using Mustard Meal

3.0 Pork Shelf Life Extension

3.1 Introduction

Part of the aim of this thesis was to investigate using mustard seed meal in food products to ultimately increase the economic viability of growing Brassicaceae seed crops for biodiesel production.  As a rotational crop, mustard provides several benefits including favorable growth requirements, pest resistance, and drought tolerance.  The oil extracted from mustard seed is suitable for producing biodiesel, but currently it is not economically viable to grow mustard for biodiesel production alone.  Seed meal is one of the byproducts following oil extraction.  Developing value-added coproducts from this seed meal may increase the market value for mustard crops. 

Mustard seed meal has many bioactive components, many of which having beneficial uses in food products.  Defatted mustard flour has historically been used in ground meat products as a water binder, preservative, and bulking agent.  It has been used in sausage and fermented meats as an antimicrobial agent and preservative.  Mustard is used as a spice and condiment in other foods as well.  As animal feed, mustard is not ideal due to its pungency and antinutritional components.  The market for mustard seed meal itself may not have potential for increase compared to strategic use of the bioactives it contains.

Brassicaceae seed meals (BSM) have conventionally been used as animal feed due to their high protein content.  Selective breeding and extraction of antinutritional components have increased their use.  In vitro digestibility of rapeseed protein (83%) is lower than that of casein (97%) partly due to less acid-induced denaturation (Savoie et al., 1988).  In humans it was found that the digestibility of rapeseed protein was between that of legume and wheat proteins (Bos et al., 2007).  In addition to its use as food, BSM proteins have been shown to have a variety of biological activities including antihypertensive, antioxidant and immunomodulating activities (Wanasundara, 2011).  This suggests that the residue remaining after antioxidant and antimicrobial extraction may be used for developing additional value-added coproducts.  

B. juncea is a mustard variety with a long history of use in food products.  Also known as brown or oriental mustard, B. juncea has a characteristic mustard-like flavor and pungency.  This pungency is due primarily to the production of allyl isothiocyanate (AIT) during processing and digestion.  Allyl isothiocyanate is a product of sinigrin hydrolysis and is a potent antibacterial compound that has been used in the food industry (Vig et al., 2009).  Sinigrin can be enzymatically hydrolyzed to four distinct aglycones: allyl isothiocyanate (AIT), allyl cyanide (AC), 1-cyano-2,3-epithiopropane (CETP), and allyl thiocyanate (ATC) (Shofran et al., 1998).  A comparison of their antibacterial and antifungal activities revealed greatest activity of AIT and no activity among sinigrin, AC, and CETP.  In a comparison of B. juncea varieties, 17 of 21 genotypes released volatiles with AIT concentrations greater than 88% total volatiles (Olivier et al., 1999).  An ethanolic extract of B. juncea was shown to inhibit the growth of several species of spoilage and indicator bacteria including E. coli, Pseudomonas fragi, and P. aeruginosa (Kanemaru and Miyamoto, 1990). The activity of the extract was similar to an equivalent concentration of AIT, suggesting that AIT is the primary bacteriocidal component of B. juncea.

In addition to its antimicrobial potential, mustard has the potential to preserve meat products through its antioxidant activity.  Extracts of Brasscaceae seed meals (BSM) have shown potent antioxidant activity resulting primarily from phenolic compounds (Kozlowska et al., 1983; Shahidi et al., 1994; Wanasundara et al., 1995).  Brasscaceae seed meals have been shown to reduce lipid oxidation in pork during storage and cooking (Brettonnet et al., 2010; Saleemi et al., 1993; Salminen et al., 2006b; Vuorela et al., 2005b).  The combined antimicrobial and antioxidant potential of B. juncea suggests the selective use of its bioactives to preserve foods may be a worthwhile investigation.

Several factors were considered during this investigation.  The goal was to find a use with realistic potential.  Earlier work with mustard and B. juncea seed meal extracts provided insight into its physical nature, composition, and flavor.  Mustard meal is allowed as an additive in foods, but the flavor is a limiting factor.  Its use has typically been limited to fresh, fermented, and cooked pork products.  Volatile oil of mustard (VOM) has been granted a generally regarded as safe (GRAS) status as an anti-spoilage and shelf life extension agent in foods, but it somewhat intensive to produce.  This extract typically contains >92% AIT, which can be difficult to apply to foods due to its volatility and toxicity.  Allyl isothiocyanate is toxic if a large amount is inhaled or consumed, having an LD50 of 151 mg/kg (Romanowski and Klenk, 2000).  In humans, AIT was found to induce short-term DNA damage, which disappeared quickly and did not affect DNA base excision repair (Charron et al., 2012).  This is less a concern when using the meal because AIT’s pungency and relatively low detection limits usually prevent consumption at overtly toxic levels (Isshiki et al., 1992).  Additionally, mustard and wasabi are used as spices in food at higher AIT levels than those used to preserve meat.  

Brassica juncea alcoholic extracts contain a variety of bioactive compounds.  The combined use of these compounds may provide benefit compared to VOM.  One particular benefit of alcoholic extraction is the ability to extract the non-volatile precursor to AIT.  Allyl isothiocyanate requires enzymatic hydrolysis, which is catalyzed by myrosinase naturally present in the seed meal when their combination is exposed to water.  Sinigrin can be preserved and extracted if myrosinase is inactivated by heat or high concentration alcohol.  Aqueous extraction has several disadvantages including the potential to hydrolyze sinigrin and cause significant swelling during extraction.  Hot aqueous extraction causes swelling of the seed meal that makes it difficult to filter and impractical for industrial scale.  Alcohol does not cause this swelling.  

Results from lab analyses indicate that maximum extraction of sinigrin is achieved using 70% alcohol at 80 oC (data not shown).  Coincidentally, these are the same extraction conditions that are conventionally used to extract antioxidants from seed meal.  Earlier work show that ultrasound treatment can increase extraction of antioxidants compared to conventional methods.  This suggests that sinigrin extraction may also be enhanced from such a treatment.  In fact, one study showed that UAE of sinigrin from B. juncea seed significantly improved extraction yield compared to conventional methods (Wang et al., 2011b).

After obtaining an extract that contains both sinigrin and antioxidants, the next step was to develop a realistic food application.  Allyl isothiocyanate has been investigated as a preservative in a variety of foods due to its antifungal and antibacterial properties, but it tends to discolor foods and impart negative flavors.  Pork was chosen as the product to study because of mustard’s historical use in pork products.  Mustard has conventionally been limited to ground meat products as it can be easily incorporated into such products. 

The predominant spoilage mechanism of retailed fresh pork is browning caused by metmyoglobin formation, putrefication caused by aerobic spoilage bacteria, rancidity caused by caused by lipid oxidation under high-oxygen MAP, acidity caused by lactic acid bacteria under vacuum or moderate oxygen packaging (Arias, 2012).  These factors are primary determinants of shelf life.  Fresh pork has relatively short display shelf life.  Factors that affect consumer decision of fresh meat products include physical appearance and aroma detection.  Loss of redness occurs due to the conversion of oxymyoglobin to metmyoglobin (Suman and Joseph, 2013).  Off-odors develop from microbial spoilage, lipid oxidation, and other deterioration processes.  One of the most effective methods to increase shelf life is modified atmosphere packaging.  

In a study comparing different atmospheric packaging on pork shelf life, 10% O2/25% CO2 fared the worst in all categories, followed by vacuum packaging (Sørheim et al., 1996).  In the absence of oxygen, combinations of CO2 and N2 preserved color equally well during storage.  Increasing the concentration of CO2 resulted in greater inhibition of psychrotropic bacteria.  However, CO2 increased the rate of drip loss.  In all packaging environments it was found that fresh flavors became less prevalent and intense during storage while unusual flavors became more prevalent and intense (Jeremiah and Gibson, 1997).  It was concluded that off-flavor development was the limiting factor in the extension of pork shelf life under MAP.

The goal of the investigation in this section was to increase the shelf life of fresh chilled pork.  This was to be accomplished by reducing spoilage while preserving favorable sensory properties.  Color is the predominant factor affecting consumer purchasing decisions of meat products.  The development of off-odors is also considered to be an acceptable measure of spoilage.  Therefore, color and odor were the two parameters that were measured during assessment of pork during storage.  

In addition to effective handling and storage procedures, modified atmosphere packaging is an effective strategy that has been widely adopted to increase shelf life.  Another common practice is the use of chemical solutions containing sodium tripolyphosphate, lactate, diacetate, nitrate and other compounds that help to preserve color, juiciness, tenderness, flavor, and shelf life.  These technologies are relatively inexpensive and quite effective.  The adoption of novel preservation technologies would thus have to exceed the performance of existing methods or potentially be used in conjunction within current systems to further prolong shelf life.

Application methods were explored based on previous scientific investigations as well as intuitive new strategies.  Application methods that were considered include mustard-based edible films, incorporation of extract into a packaging film, direct application of extract onto the meat surface, and strategic hydrolysis of sinigrin into AIT that could fill the package headspace.  

One potential application of mustard is edible films (Hendrix et al., 2012).  Such a film was able to reduce the rate of lipid oxidation and production of off odors compared to uncoated samples (Kim et al., 2012).  The same type of mustard-based film was also shown to have antimicrobial activity against Listeria monocytogenes (Lee et al., 2012).  An AIT-rich horseradish extract was shown to inhibit lipid oxidation of pork when applied to a plastic packaging film (Jung et al., 2009).  A film containing mustard extract showed antimicrobial activity in sausage (Lara-Lledó et al., 2012).  These studies support the potential use of mustard-based film and applying mustard extract as a coating on pork to reduce spoilage.

One unique advantage of using a mustard-based film with additional B. juncea extract is the potential for controlled AIT production.  The enzyme that hydrolyzes sinigrin to AIT is not effectively extracted.  Mustard-based films can be developed using methods that preserve active enzyme.  Brassica juncea extract can then be incorporated onto the film, creating a product that will produce AIT upon contact with water.  When this combination is applied to the surface of meat, AIT will be produced and may work synergistically with other compounds in mustard to reduce spoilage.  Alternatively, this same dry extract-enzyme combination could be incorporated into the meat absorbent pads that absorb purge.  When water from the purge combines with sinigrin and myrosinase, AIT will be produced and fill the headspace.  This would allow meat pads to continue to be used as usual and only produce AIT when exposed to water.

Introducing AIT into the headspace of food packages has been investigated with a variety of foods.  However, its in vitro antimicrobial activity has not always translated to similar activity in food products.  Greater concentrations of AIT were required to inhibit growth of spoilage bacteria in pork compared to in vitro growth medium (Schirmer and Langsrud, 2010).  AIT can react with meat proteins, and this may affect its antimicrobial activity.  It can also react with free amino groups of lysine and arginine residues.  Allyl isothiocyanate can cleave the cystine disulfide bonds and form polymers (Kawakishi and Kaneko, 1987).  It has been shown to react with glutathione and cysteine naturally present in meat, reducing the antimicrobial potential (Luciano et al., 2008).  This reaction readily occurs at storage conditions and may partly explain the reduction antimicrobial activity in meat products compared to cultured media.  Nevertheless, AIT in headspace has significant antibacterial activity in meat at sufficient concentrations (Nadarajah et al., 2005b).

Despite its effective, there has been limited development of antimicrobial systems that release AIT into food packaging headspace (Coma, 2008).  Exploration of patent applications returned a single company that has applied for numerous patents related to releasing AIT in packaging headspace to preserve foods.  WasaOuro® by Green Cross Co. (Japan) is a commercial product available in Japan that consists of an insert with controlled AIT-release that can be placed in food packages.  This technology presents a convenient method to apply AIT to a variety of foods and is similar to the techniques explored in this section.

3.2 Materials & Methods

3.2.1 Materials

All experiments were performed using meal remaining after cold pressing of B. juncea ‘Pacific Gold’ and Sinapis alba ‘IdaGold’ seeds in a press on the University of Idaho campus (Moscow, ID) (Brown et al., 1998, 2004)  Approximately 90% of the oil was extracted from the seeds using a mechanical seed crusher (Borek and Morra, 2005; Peterson et al., 1983).  The meal consisted of irregularly shaped flakes ranging from approximately 1 mm to 3 cm (Hendrix et al., 2012).  Allyl isothiocyanate ≥ 95% was obtained from Sigma-Aldrich Co. (St. Louis, MO, USA).

3.2.2 Meat Selection

Fresh pork (thin-sliced loin chops and 20%-fat ground pork) was purchased from local grocery markets three days before the “best before” date.  The chops were in an overlapped arrangement in the display package.  When chops were separated there was slight discoloration at the contact surfaces.  To restore color, chops were individually wrapped and stored at 4°C overnight.  This allowed for a uniform distribution of color on the surfaces of all pork samples.  Similarly, ground pork was formed into patties and allowed stored at 4°C overnight to establish a consistent starting color of all patties.

3.2.3 Extract Preparation

Brassica juncea seed meal was extracted with 70% ethanol using a scaled-up method based on conventional extraction procedure.  Two hundred grams of meal were placed in a 1000-mL Erlenmeyer flask to which 800 mL of solvent was added.  The mixture was heated in a water bath maintained at 80 °C for 60 min.  The mixture was then filtered through a 30 mesh stainless steel strainer and the solid residue was extracted four more times under the same conditions.  The extract was concentrated using a rotary evaporator and stored at 4 °C.  Extract was analyzed for sinapine and sinigrin content by HPLC and it was determined to contain 300 µM sinigrin and 10 µM sinapine.

3.2.4 Film Preparation

Films were prepared according to the procedure described by Kim et al. (2012).  Sinapis alba meal was chosen to develop the film both because it has been used previously and because B. juncea meal caused excessive foaming during homogenization (Hendrix et al., 2012).  Briefly, 14.0 g ground defatted s. alba meal was dispersed in 184 g deionized water for 30 min and the mixture was homogenized at 20,000 rpm for 5 min.  The homogenate was mixed with 2.0 g of glycerol and stirred for 30 min.  Polysorbate-20 (0.14 g) was added, and the solution was homogenized at 20,000 rpm for 5 min.  The mixture was degassed under vacuum.  Films were cast by pipetting mixture onto PTFE plates (12.5 cm dia) resting on a leveled surface and dried at room temperature for 2 days.  The amount of the mixture (34.5 g) pipetted for each film was selected to produce a 0.2 mm thick film.  Dried films were peeled from the casting plate and stored in polypropylene containers.

An additional batch of films was prepared and incorporated with additional B. juncea extract.  After films were dried on casting plates, 5.0 mL B. juncea extract was pipetted onto the plates and dried at 60 °C and 15 % relative humidity for 1 hour.  These conditions were used to accelerate drying and to limit the extent of hydrolysis of sinigrin in the extract without deactivating myrosinase.  This resulting films contained active myrosinase that can hydrolyze sinigrin to AIT.  The film incorporated with additional extract thus creates an edible film that can be applied to the surface of foods to produce a volatile antimicrobial upon contact with water.

3.2.5 Film Application

This first experiment explored the potential use of films to prolong the shelf life of pork.  Ground pork was chosen because lipid oxidation was being considered as an additional assay to measure shelf life.  Mustard has also historically been used as an additive in ground pork and sausage products, thus residual flavors in pork may be more acceptable than in other products.  Brassica juncea extract is rich in antioxidants as well as antimicrobial precursors.  The direct application of films incorporated with extract may confer both antioxidant and antimicrobial protection, and ground pork with its high fat content could be used as a model to test this.  

Ground pork was formed into 100 g patties at a thickness of approximately 1.5 cm.  Two films were used for each patty and applied to the top and bottom surfaces so the entire patty surface was in contact with the film.  Both supplemented and non-supplemented films were tested.  An additional treatment group was included that incorporated 10.0 mL extract into each patty as it was initially being formed.  Samples were prepared in duplicate.  Samples were individually stored in Ziploc® freezer bags at 10 °C for 6 weeks.  Samples were taken in 1-week intervals and assessed for quality. 

3.2.6 Direct Extract Application

The next experiment explored the direct application of extract onto the surface of pork without using films.  Pork chops were chosen for this experiment instead of ground pork.  Consumer demand for fresh pork is higher for intact meat than ground meat.  To maximize shelf life of fresh pork, such as during shipment overseas, the market would be greater for intact pork than ground pork.  Additionally, intact meat has a longer shelf life than ground meat.  With the goal being to maximize shelf life, it was considered worthwhile to continue experimentation using intact meat.  Pork loin chop is a popular and common cut of meat and was considered suitable for this experiment.  

Pork chops were cut into 50 g sections at a thickness of approximately 1.0 cm.  Immediately prior to use, B. juncea extract was supplemented with mustard flour at an extract-to-meal ratio of 50:1 (v/w).  Chops were dipped into this solution and allowed to dry at 40°C for 5 min.  This was repeated twice more to provide an even coating that totaled about 5.0 mL of original extract per sample.  Samples were prepared in duplicate.  Samples were individually stored in Ziploc® freezer bags at 10°C for 3 weeks.  Samples were taken in 1-week intervals and assessed for quality.

3.2.7 AIT Headspace Treatment

The next experiment explored the technique of hydrolyzing sinigrin to AIT within the packaging headspace.  Brassica juncea extract contains intact sinigrin, the precursor to the volatile antimicrobial AIT.  Direct application of extract onto the surface of meat has been disadvantageous by negatively affecting color and flavor.  Producing AIT within the headspace of the food package may provide antimicrobial activity without these negative effects.

It was at this time that modified atmosphere packaging (MAP) was considered.  Modified atmosphere packaging effectively suppresses the growth of pathogenic and spoilage bacteria in meat as well as reduces the rate of lipid and protein oxidation.  In the absence of oxygen, combinations of CO2 and N2 are able to reduce spoilage and preserve meat color during storage.  In pork products under MAP, off-flavor development is typically the limiting factor in the extension of shelf life.  Greater concentration of CO2 results in greater inhibition of psychrotropic bacteria.  However, CO2 increases the rate of drip loss.  In order to test whether AIT and other mustard-volatiles could further suppress the development of off-odors, 100% CO2 was chosen for atmospheric conditions during storage.

Pork chops were cut into 50 g sections at a thickness of approximately 1.0 cm.  One gram of mustard flour was added to 50 mL B. juncea extract and placed in an open container alongside each pork sample within dual-layered Ziploc® freezer bags.  The air was removed from the packages using vacuum and was replaced with 100% CO2.  To simulate accelerated storage and provide proof of concept, samples were prepared in duplicate and stored at 15°C for 3 weeks.  Samples were taken in 1-week intervals and assessed for quality.

3.2.8 Active AIT-CO2-MAP 

The final experiment was based on the results of the previous preliminary experiments.  Briefly, the objective and subjective results of the preliminary experiments showed that volatiles released from high amounts of B. juncea could inhibit the production of off-odors at the expense of color.  This is consistent with results of other studies using AIT to reduce microbial growth in meat (Delaquis et al., 1999; Shin et al., 2010).  There appears to be an optimal range of AIT levels that can be used to inhibit microbial growth without significantly affecting meat color.  There are few studies that combine the use of AIT and MAP to prolong shelf life of meat products.  This final experiment tested the use of volatile released from B. juncea extract in combination with MAP to reduce off-odor development in pork while preserving color.

Pork chops were cut into 50 g sections at a thickness of approximately 1.0 cm.  Brassica juncea extract (12.0 mL) was mixed with 240 mg mustard flour and placed in a Ziploc® snack bag, composed of thin low-density polyethylene (LDPE).  This release-system was developed to simulate a device that could produce AIT as desired.  Low-density polyethylene is permeable to AIT, the permeation rate of which depends on the thickness and exact composition of the plastic.  The combination of sinigrin, myrosinase, and water within this bag would produce AIT that could permeate through the bag into the food package and be retained within an impermeable outer package.  The material chosen as outer packaging was FoodSaver® (Tilia, San Francisco, CA) nylon-polyethylene vacuum bags composed of several different polymer layers that are very impermeable to both AIT and oxygen.  One each of a pork sample, AIT-release package, and 100 cc oxygen absorber (Sorbent Systems, Los Angeles, CA) were packaged together in a FoodSaver® vacuum bag.  The air was removed from the packages using vacuum and was replaced with 100% CO2.  

Two other treatments groups were prepared.  One group was treated with 180 µL pure AIT.  This level of AIT contains the molar equivalent as that of sinigrin in 12.0 mL B. juncea extract.  This is the maximum expected amount of AIT that could be produced from the hydrolysis of sinigrin in the extract.  If there develops less AIT in the headspace of the extract treatment, it may be explained by incomplete hydrolysis of sinigrin and volatilization of AIT from the extract.  The final treatment group used half the level of extract as the initial treatment (6.0 mL extract).  Samples were prepared in duplicate and stored at 10°C for 30 d.  Samples were taken in 5-d intervals and measured for color and odor.

3.2.9 Color Analysis

The surface color of pork samples was measured using a Konica Minolta CR-400 Chroma Meter (Konica Minolta Sensing, INC. Osaka, Japan) calibrated with a standard white plate (Y = 94.00, x = 0.3158, y = 0.3322).  Readings of CIE L* (Lightness), a* (green chromaticity) and b* (yellow chromaticity) coordinates were recorded for each measurement.  Reported values are the mean ± standard deviation of ten measurements.  The color of pork samples was not uniformly distributed and measurements were taken throughout the surface to ensure the variability of color was accounted for.  One-way analysis of variance and Bonferroni t-tests were performed to assess the effects of treatment on color measurements (Statistical Analysis Software, SAS/STAT version 9.2).

3.2.10 Odor Analysis

Odor analysis was conducted using an electronic nose (4100 vapor analysis system, Electronic Sensor Technology, New Bury Park, USA).   The zNose™ is a portable bench top gas chromatography system for field or laboratory use.  It uses a single, uncoated, high quartz surface acoustic wave (SAW) sensor consisting of an uncoated 500 MHz acoustic resonator bonded to a Peltier thermoelectric heat pump.  Advantages of this system include high sensitivity, simple signal measurement and handling, and low power requirements (Nurjuliana et al., 2011).

Five grams of each pork sample was minced and sealed into 40mL glass vials.  After vials were allowed to equilibrate at room temperature, vapor was pumped for 5s into the electronic nose using a sampling needle through a septum.  Within the unit, vapor was concentrated in a trap (inlet 200 °C), which was rapidly heated, focusing the releasing vapors onto a low-temperature (40 °C) capillary column.  The column temperature was programmed to linearly heat from 40 to 180 °C at a rate of 10 °C/s.  This causes the different components in the vapor to release as they travel through the column and land on the SAW sensor (60 °C).  When volatiles are adsorbed on the sensor surface, the frequency of the SAW will be change and affect the detection signal, allowing identification of the compounds.  The flow rate of helium was fixed at 3.0 mL/min.  The total cycle time per sample was 40 s.  Measurement vials were prepared in duplicate from each sample.

3.3 Results & Discussion

3.3.1 Film Application

Films were applied to pork samples so that the entire surface was in contact with the film.  The uncoated films initially had a yellow translucent appearance (Illustration 3.1).  The extract-coated films appeared thicker, darker, and browner than uncoated films.  Coated films were also subjectively less plastic, less elastic, and more brittle.  During larger scale film production with accelerated drying (refractance window drying), films often cracked and dried unevenly.  Additionally, the heat provided to accelerate drying resulted in inactivation of the myrosinase enzyme needed to catalyze the sinigrin in the B. juncea extract to AIT.  These are some of the challenges facing large-scale development of mustard meal-based edible films. 

Both films were readily water soluble and quickly disintegrated on the meat surface.  The resulting paste did not provide a uniform vapor barrier and could easily be moved and transferred to adjacent contacting surfaces.  During storage, the paste conferred an unappealing color to the meat (Illustration 3.2).  The uncoated film conferred a pale yellowish color whereas the coated film conferred a dark brown color.  Extract incorporated within the meat patties resulted in browning as well.

Compared to the control, all three treatments resulted in inhibition of off-odor development during storage.  However, uncoated samples developed visible surface mold growth by 4 weeks.  After 6 weeks, the samples in the control group had decayed to the point that the meat was no longer solid.  No treatment group resulted in this degree of decay.  Nevertheless, samples in the two groups treated with uncoated films and extract incorporation had clearly spoiled.  What was interesting was that after 6 weeks, the samples treated with coated films had no obvious signs of decay.  They were similar in appearance and aroma as samples after week 1, and there was minimal off-odor.  There was a noticeable pungent aroma, which suggested the presence of AIT.  The texture was also tougher and the surface was dryer than fresh untreated pork.  This may have been due to the surface drying effect of film application and the interaction of AIT with meat proteins.

The results of this preliminary experiment suggest that mustard meal-based films are unsuitable for commercial application.  They have poor mechanical and gaseous properties and there are difficulties during scale-up production.  Film application on fresh pork appeared to reduce spoilage, particularly by extract-coated films, but resulted in an undesirable appearance and texture.  

3.3.2 Direct Extract Application

Brassica juncea extract was applied onto pork chop surfaces by dipping them directly into the extract.  Each coating dried quickly resulting in a uniform coating that could allow additional coatings as desired.  It was found that increasing the number of coatings led to a more orange brown appearance.  This was expected as the extract itself has an orange brown color.  The results after storage were similar to those of film-coated pork.  At week 3, the untreated control samples had lost their desirable pink color and appeared tan and gray (Illustration 3.3).  The coated pork samples were darker and more orange and brown in color.  

Similar to previous experiment, after week 3, the coated samples had no obvious signs of decay.  They were similar in appearance and aroma as samples after week 1, and there was minimal off-odor.  There was still a noticeable pungent aroma, which suggested the presence of AIT.  The texture was also noticeably tougher, possibly due to the interaction of AIT with meat proteins.

The physical appearance of the treated samples in this experiment more closely resembled fresh pork than the film-coated samples of the previous experiment.  There was no paste-like coating that masked the pork surface.  Nevertheless, color was still adversely affected.  The results of this experiment, along with those of the previous experiment, suggest that inhibition of spoilage can be achieved by surface application of B. juncea extract but at the detriment of color.  Further experimentation focused on indirect application of extract in hopes that color can be preserved while still providing protection from spoilage. 

3.3.3 AIT Headspace Treatment 

Pork chops were stored in 100% CO2 MAP at 15 °C for up to 3 weeks.  The headspace of the treatment groups had additional volatiles that were produce during the enzymatic hydrolysis of glucosinolates in B. juncea extracts.  At the end of week 3, samples in the control group maintained their desirable pink color (Illustration 3.4).  However, they had begun to develop off-odors.  These off-odors were not readily detectable in the treatment groups.  Instead, there was a pungent aroma, which suggested the presence of AIT in the headspace.  However, there was dramatic darkening and graying of the treatment groups during storage (Illustration 3.4).  

Other studies using volatilized AIT to inhibit spoilage in meat have not observed this dramatic color change (Delaquis et al., 1999; Shin et al., 2010).  There are several possibilities that may explain this discrepancy.  Brassica juncea extract contains glucosinolates other than sinigrin.  These are usually present in low concentrations, but they may still be hydrolyzed into volatile isothiocyanates.  These additional isothiocyanates along with AIT and other odorous compounds may be interacting with meat differently than isolated AIT.  The extract was not in direct contact with the meat surface, and this may explain the lack of orange color compared to previous experiments.  It was undeterminable in the previous experiments if the color change was due to the color of the extract itself, the interaction of extract compounds with the meat, or their combination.

Another possibility is that the use of Ziploc® bags may have provided an insufficient gaseous barrier to preserve the 100% CO2 atmosphere.  There was noticeably less headspace after storage than at the initial package preparation, but this may have been partly due to the absorption of carbon dioxide into the pork itself.  The headspace composition may have affected the interaction of AIT with the meat surface and thus the color change.  The preservation of color in the control group suggests that the headspace atmosphere itself did not cause the discoloration.

Other possibilities include the presence of unknown compounds in the meat, the temperature during storage, and the high level of extract used relative to the mass of pork.  There are other possible explanations, and it was unclear which factors should be addressed in later experiments.  Factors that could readily be addressed include using a different packaging material, using an oxygen absorbent in the package, using less extract relative to the amount of meat, and enclosing the extract in a container that is permeable to AIT but possibly impermeable to other volatiles.  The resulting treatment may reduce the detrimental effect B. juncea has on color while preserving its antimicrobial activity.

Modified atmosphere packaging using 100% CO2 is effective in preserving color during storage and suggests the limiting factor of shelf life may be spoilage that leads to off-odors.  Treatment with B. juncea has consistently reduced the obvious development of off-odors.  Brassica juncea volatiles combined with MAP may be effective in extending shelf life if there is minimal detrimental effect on color. 

3.3.4 Active AIT-CO2-MAP 

Modified atmosphere packaging alone is sufficient to inhibit spoilage and preserve color.  The hypothesis is that addition of B. juncea volatiles in packaging headspace can increase the effectiveness of MAP.  Other studies have used AIT to preserve meat with minimal discoloration (Delaquis et al., 1999; Shin et al., 2010).  It could be predicted that AIT released from B. juncea would have similar effects.  To control for the previously mentioned factors, several techniques were implemented in this final experiment.

Packaging material with low gaseous permeability was used.  An oxygen absorbent was included in each package to minimize oxygen content in the headspace.  The extract mixture was contained in a thin LDPE bag through which AIT could permeate into the headspace while potentially leaving other compounds entrapped in the bag.  A photograph of the resulting package appears in Illustration 3.5.  

There were three treatment groups in addition to the untreated control.  One treatment group used 12.0 mL extract per package, which contained 710 µg sinigrin.  The second treatment used half this amount (6.0 mL extract).  The final treatment used pure 180 µL pure AIT, the molar equivalent of sinigrin contained in 12.0 mL extract.  Samples were stored at 10°C for 30 d.  Samples were taken in 5-d intervals and measured for color and odor.

3.3.5 Color

Pork surface color measurements for CIE L* a* and b* values appear in Figure 3.1, Figure 3.2 and Figure 3.3, respectively.  Lightness of the control group remained constant throughout storage (p > 0.05).  The three treatments experienced small but significant increases in lightness during storage compared to initial readings and those of the control group at day 30 (p < 0.05).  

Green chromaticity decreased in all groups by day 10 (p < 0.05).  Among all groups, values were similar at day 30 as day 10 (p > 0.05).  Green chromaticity (a*) is a coordinate between magenta (positive values) and green (negative values).  The observed decrease in green chromaticity indicates a slight loss of redness during storage.  However, this loss of redness only occurred during the first 10 days.  There was no difference among treatment groups or the control group at day 10 or day 30, suggesting that the initial loss of redness was caused primarily by the MAP, not the AIT.

Yellow chromaticity followed a similar trend.  There was an initial decrease in values by day 5 among all groups, which was greater in the three treatment groups than the control (p < 0.05).  From day 5 to day 30, values in the control and two extract treatment groups remained the same (p > 0.05), whereas values in the pure AIT treatment increased (p < 0.05).  Throughout the storage period, values of the two extract treatment groups were similar and were less than the control (p < 0.05).  Yellow chromaticity (b*) is a coordinate between yellow (positive values) and blue (negative values).  The observed decrease in yellow chromaticity indicates a slight loss of yellowness during storage.  The sudden initial decrease in yellowness in all groups suggests it was caused primarily by the MAP.  The increase in yellowness in the AIT treatment group from day 5 to day 30 is consistent with other studies that have used AIT at high levels (Shin et al., 2010).  This increase was not observed in the extract treatment groups, suggesting a possible benefit of extract treatment if excessive yellowness is undesirable.

To illustrate the appearance of the treatment groups, photographs of samples from day 10 through day 25 appear in Illustration 3.6.  There is little obvious difference among treatment samples and minimal trends of color change during storage.  To further compare the color of the groups, the L*a*b* measurement data were used to generate color swatches that appear in Illustration 3.7.  Again, there are no obvious differences or trends among treatments and differed slightly compared to the initial fresh pork.  These results show that this protocol combining MAP with AIT did not detrimentally affect the appearance of pork compared to previous preliminary experiments.

3.3.6 Odor

Electronic nose systems have been used to identify a broad range of odorants in pork and other meat products (Ghasemi-Varnamkhasti et al., 2009; Nurjuliana et al., 2011; Tian et al., 2011).  The goal of this analysis was to qualitatively show the appearance of dominant odors of stored pork samples.  The premise was that odors appearing during storage of untreated samples represent off-odors resulting from spoilage.  Therefore, the lack of such odors in treated samples would suggest a reduction in off-odor development and thus spoilage.  The hypothesis was that the volatiles released from the extract would inhibit spoilage and the production of off-odors.  A sufficient database to identify peaks was not available, and analysis was based on comparisons to standards and other associations.  Low sensitivity settings were used to identify the most abundant odors and make clear comparison among sample odor profiles.  

Odor analysis was conducted using a zNose™ portable bench top gas chromatography system.  Chromatograms generated from this system are graphical displays of the frequency change derived with respect to time.  Each peak corresponds to a specific volatile compound whose retention time is specific to the column and run method.  The area under the peak is correlated to the compound concentration and is expressed in counts (Ct).  Samples were analyzed for the presence of AIT, mustard volatiles, and odorants associated with spoilage.  

The chromatogram of the low-level extract treatment (6.0 mL B. juncea extract per package) at day 5 appears in Figure 3.4.  There were two primary peaks observed.  One peak is believed to be AIT (labeled A) as it had the same retention time as pure AIT.  This was expected as AIT is the primary isothiocyanate produced during the hydrolysis of glucosinolates in B. juncea extract.  These samples smelled somewhat pungent, which is a characteristic of AIT, and this analysis confirmed the presence of AIT.  It also confirmed the production and permeation of AIT from the extract through the ethylene bag.  The second peak (labeled M) is believed to be an aroma of the B. juncea extract itself.  Analysis of B. juncea extract before and after analysis revealed the same peak with the same peak height, suggesting it was not affected by enzyme treatment.

The chromatogram of the control sample at day 30 appears in Figure 3.5.  Analysis of control samples at day 0 and day 5 revealed no identifiable peaks.  It is believed that the peaks that appeared after storage represent off-odors associated with spoilage.  The largest peak (labeled S) was used in comparisons to treatment samples.

The chromatogram of the high-level extract treatment at day 30 appears in Figure 3.6.  The M and A peaks are still present, but the S peak appears shorter than in the control samples.  If the peaks observed in the control samples are considered off-odors, there appears to be less off-odor in the extract treatment group, potentially indicating less spoilage.  This suggests that extract treatment reduced the rate of spoilage and off-odor development.  

The chromatogram of the low-level extract treatment at day 30 appears in Figure 3.7.  The M peak is still present; however, the A peak is smaller.  Analysis of samples throughout the storage period showed a decreasing A peak height, suggesting AIT was decreased during storage.  This may have been caused by interaction of AIT with bacteria and meat components.  The S peak also appeared smaller compared to the control, suggesting there was inhibition of off-odor development.  These samples smelled reminiscent of mustard, but were not obviously spoiled or pungent.

If these suspicions of behavior are true, then this is a significant finding.  One of the limiting factors of using AIT in the food industry is its pungency.  If using B. juncea extract to release a moderate level of AIT in the MAP headspace of meat products can reduce spoilage while insignificantly affecting color, there may be potential market demand for the technology.  It also highlights the effectiveness of combining preservation techniques.

Another notable finding from odor analysis was that pure AIT treatment resulted in a much larger A peak in samples than either of the extract treatments.  This suggests incomplete production and permeation of AIT in the package.  This higher level of AIT compared to extract treatments might explain the increase in yellow chromaticity measured from day 5 to day 30.

3.3.7 Discussion

The results of this experiment have several implications.  It is already known in the food industry that MAP is a very effective technique to reduce spoilage and prolong shelf life.  It is also currently used in retail operations in such products as ground meat to preserve freshness during centralized production and distribution.  Allyl isothiocyanate is an effective antimicrobial that inhibits growth of bacteria and fungi.  It has been used in meat products to inhibit the growth of pathogenic and spoilage bacteria.  However, at high levels it can adversely affect meat color and flavor.  The use of moderate levels of AIT in combination with MAP may synergistically extend the shelf life beyond either technology alone.  Such a combination has been studied in chicken, catfish, and cheese (Pang et al., 2013; Shin et al., 2010; Winther and Nielsen, 2006).  In all cases the combination of AIT and MAP significantly prolonged the shelf life of these foods.  One major consideration in the addition of AIT to MAP is the delivery system.

The use of B. juncea extract, or isolated sinigrin, may offer advantages compared to isolated AIT.  Pure AIT is dangerous and harmful at high exposure levels.  Brassica juncea extract and sinigrin do not have this concern.  When combined with myrosinase, sinigrin can be hydrolyzed at a predetermined time, such as when the release system is placed in a package with meat.  As long as they remain dry and stored in low humidity environments, there won’t be AIT production.  This allows for flexible storage and application requirements.

Translating this to a marketable product, if sinigrin and myrosinase were to be combined and incorporated in meat absorbent pads placed beneath meat samples to absorb purge, the water from the purge would initiate AIT production.  Such a product would not require significant change in the manner in which meat is packaged.  In operations that use MAP, the absorbent pads could simply replace conventional pads.  Alternatively, a product could be developed that contained sinigrin, myrosinase, and water within a small permeable container.  At least one component would be isolated from the others, such as water being contained in an internally breakable plastic container.  When broken, the components would mix and AIT would be produced and permeate through the external barrier.

Such products have the potential to reduce spoilage and preserve food products.  As a major source of sinigrin and myrosinase, B. juncea seed meal can be used for such purposes.  This may lead to an increase in the price of B. juncea crops.  The key determinant of future industry adoption of such technologies would be whether the benefit they provide exceeds the expense, inconvenience, and acceptance of their implementation.


Contact me for references. The reference list for my thesis is about 20 pages long

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