The Effect of Combining Propionic and Ascorbic Acidon the Keeping Qualities of Fresh Minced Pork during

StorageSharon K. Ogden, Andrew J. Taylor, Christine E. R. Dodd, Isabel Guerrero, Hector Escalona Buendia and

Francisco Gallardo

S. K. Ogden, A. J. Taylor, C. E. R. Dodd: Department of Applied Biochemistry and Food Science, University ofNottingham, Sutton Bonington Campus, Loughborough LE12 5RD (U.K.)

I. Guerrero, H. E. Buendia, F. Gallardo: Department of Biotechnology, Universidad Autonoma Metropolitana,Iztapalapa, Apartado Postal 55-535 C.P. 09340, Mexico D.F (Mexico)

(Received May 8, 1995; accepted June 27, 1995)

In an attempt to improve the bacteriological quality of raw pork Longissimus dorsi mince meat, without adversely affecting theodour, colour and texture, the meat surface was treated with individual solutions of propionic acid and ascorbic acid, and acombination of the two acids, at concentrations that had a preservative effect. A water-treated sample was used as the control.Bacterial colonization was determined over a 13 d refrigerated (4 °C) storage period. Propionic acid at 0.133 mol/L reduced thepseudomonad count by 3 log10 cfu/g over this period, whereas 0.41 mol/L propionic acid reduced pseudomonad counts by 8log10cfu/g. Combinations of ascorbic and propionic acid solutions were effective in reducing the microbial load of the mincedpork.Headspace volatiles from untreated and treated minced pork were trapped on Tenax and analysed by GC-MS. Greater amountsof lipid oxidation were found in all acid-treated meats (apart from ascorbic) relative to the water-treated control sample.Meat colour was best preserved when ascorbic acid was present. Minced pork treated with solutions of propionic acid showedsurface bleaching and an increase in lipid oxidation volatiles when compared with the control. All acid treatments decreased thewater-holding capacity of the meat.

©1996 Academic Press Limited

Introduction

Microbial growth in fresh meat is one of the primaryfactors associated with meat quality reduction, spoilageand economic loss. During conventional slaughterprocedures and further processing necessary to preparemeat for consumption, microorganisms are introducedinto and onto carcasses (1,2).Increased consumption of meat in many less-developedcountries is limited by a number of factors, for examplethe difficulties and expense of establishing suitable coldchains for the storage and distribution of meat andmeat products. Foods that can be stored withoutrefrigeration are of special interest in developingcountries.In order to overcome these problems, there is a needfor low-cost preservation systems to provide meat thathas microbiological and chemical stability at tropicalambient temperatures. The use of food-grade organicacids to reduce the number of microorganisms on meatsurfaces is well documented (3–6), although all experi-ments have been carried out at 4°C. Organic acid sprayshave recently been allowed by law (in Belgium,Australia and the Federal Republic of Germany) asfresh meat decontaminants. It is generally accepted that

it is the lipophilic, undissociated acid molecule that isresponsible for the preservation effect of organic acids(7,8). The extent of the antimicrobial action of anorganic acid depends upon its pK, the pH of theexternal medium and the buffering capacity of the meat(9). It has been reported (10) that combinations of twoor more acids are more effective than treatment withindividual acids. Previous work in our laboratory (11)has studied the use of dilute solutions of lactic andpropionic acids over a range of concentrations thathave been found to control spoilage bacteria at 4°Cthrough an intermediate bacteriological effect and aresidual bacteriostatic action. However, concentrationsgreater than 0.19 mol/L lactic acid or 0.27 mol/Lpropionic acid resulted in unacceptable tissue dis-colouration, and off-odours, due primarily to lipidoxidation volatiles. Thus, acid treatment of meatsincreases lipid oxidation, which is a major factorinfluencing the quality of foods, especially meatproducts.Very few reports have appeared in the literature thatrelate the effects of organic acids to the quality of meat.The objective of this work was to study the effect ofascorbic acid alone and in combination with propionicacid on the microbiological quality, texture, odour

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Table 1 The concentrations and types of acid used

Acid type Acid concentration (mol/L)

Propionic 0.136 and 0.410

Ascorbic 0.057 mol/L

Propionic and ascorbic 0.102 propionic, 0.014 ascorbic0.204 propionic, 0.028 ascorbic0.306 propionic, 0.043 ascorbic0.136 propionic, 0.057 ascorbic

profile and colour of refrigerated meat (4°C) storedover 13 d. Several research groups (12) have reportedthe antioxidant effects of ascorbic acid. It couldtherefore have the potential to inhibit lipid as well aspigment oxidation. If the technique is successful duringmeat storage at 4°C, its applicability at higher tem-peratures will be studied, and the effect that low pHvalues will have on sensory properties and consumeracceptability will be investigated by washing excess acidoff prior to eating.

Materials and Methods

Meat samplesPost-rigor muscles from boars of about 6-months-oldthat had been fed on the same diet were obtained fromthe university slaughterhouse and stored at 4°C. L-as-corbic acid, obtained from Sigma (Poole, Dorset, U.K.),propionic acid (purity 990 g/kg after hydrolysis) andsodium chloride were obtained from Fisons (Loughbor-ough, Leicestershire, U.K.), article numbers A-7506,P7560/PB08 and S/3160/53, respectively. Plate CountAgar (0479-17) and Pseudomonas Isolation Agar(0927-17-1) were products of Difco laboratories (Sur-rey, U.K.).Meat batches (7 batches; 3.125 kg/batch) were preparedfrom 25 kg minced L.dorsi. Each batch was subdividedinto 300 g batches after it had been mixed for 5 min atspeed 1 in a Kenwood-Peerless Planetary mixer (Bir-mingham, U.K.), with solutions of propionic or pro-pionic and ascorbic acid at the concentrations shown inTable 1.The ratio of acid solution to meat was 0.44 L/kg meat.Excess acid solution was drained from the meatportions, which were then heat sealed into bags andstored at 4 to 6°C for a maximum of 13 d, samplingstarting on day 1.

Microbial analysisThe bacteriostatic and bactericidal effects of the acidtreatments were determined by total aerobic counts(TACs) on plate count agar and Pseudomonas counts.Meat (2 g) from each acid treatment was added tomaxium recovery diluent (MRD; 20 mL; pH 7.0 ± 0.2)and shaken. Subsequent dilutions were made in sterileMRD. Counts were determined by spread plating 0.1mL of sample in duplicate and incubating at 32°Caerobically for 48 h before counting (13). Counts wereestimated from the water-treated control and the acid-

treated samples from 0 to 13 d refrigerated storage, andwere recorded in colony forming units per gram ofmeat (cfu/g).

Collection and analysis of volatilesA Tenax trap (CHIS, SGE, Milton Keynes, U.K.) washeld in the headspace of the bag containing the mincedmeat and volatiles were collected for a period of 7 minusing a vacuum pump (flow rate 40 cm3 air per min). Anexternal standard (benzaldehyde in methanol (2 µL))was added to the trap under vacuum to compensate forany instrumental or chromatographic variation thatmight occur during the storage and analysis period. Thepeak areas of the volatiles in the meat were expressedrelative to the peak area of the external standard, thusreducing the standard deviation (sx) between replicates.The volatiles were analysed on a VG MD 800 bench topmass spectrometer (Fisons Scientific, Manchester,U.K.) connected to a Hewlett Packard 5890 Series IIgas chromatograph fitted with a headspace injector(CHIS, SGE). The meat volatiles were desorbed fromthe traps at 240°C (column head pressure 124 kPa,carrier gas helium) and transferred to the column (25 m3 0.22 mm, i.d. BP-1; 1.0 µm film thickness; SGE) for2 min. During desorption, a 400 mm region of thecolumn was cooled in liquid nitrogen to cryotrap thevolatiles. Following desorption, the sample was chro-matographed from 30 to 112°C at a ramp rate of4°C/min and then from 112 to 240°C at a ramp rate of7°C/min after a 2 min delay. Volatiles were detected onthe MD 800 and quantified by single-ion monitoring(SIM) of characteristic ions. The relative amounts wereexpressed as peak areas, which were corrected byreference to the external standard.

Colour measurement systemColour measurements were made on the surface ofchilled (4°C) minced meat samples using a HunterlabReflectance Tristimulus Colorimeter (Model 5330),which was calibrated using the white and grey standardtiles (standard number SN C5330, assigned values:x = 48.15, y = 51.34, z = 56.28 for the grey standard,and x = 82.78, y = 87.63, z = 95.08 for the white stan-dard). Minced meat (10 g) was wrapped in cling-film(European Plastic Film Manufacturers Association,U.K.) and placed at the specimen light port. Hunter L(lightness), a (redness) and b (yellowness) values wereobtained, for three replicates, and from these, hueangles (Tan–1 (b/a)) and chroma (p √a2 + b2) werecalculated.

Water-holding capacity (WHC)The WHC was obtained by using a modification of thecentrifugation method (15). Minced meat (5 g) wasplaced in a test tube, NaCl (8 mL; 0.6 mol/L) was addedand the samples stirred with a glass rod for 1 min. Thetubes were placed in an ice bath for 30 min andoccasionally stirred. The samples were stirred for 1 min

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before being centrifuged at 10,000 rpm for 30 min. Thesupernatant was decanted into a 5 mL graduatedcylinder, and the volume of 0.6 mol/L NaCl retainedwas recorded for the three replicate samples and thenexpressed as the WHC/g on a dry weight basis (typicalpercent coefficient of variation [sx 3 100/mean] being2.8%).

pH measurementSamples of meat (10 g) were mixed with 100 mLdistilled water and the pH determined after approx. 1hour using a CD 620 digital pH meter (Fisons,Loughborough, U.K.) standardized at pH 4 and 7 usingcommercial calibration solutions (Fisons, Loughbor-ough, U.K.).

Results and Discussion

Effects of organic acids on pH and microbialcolonization of minced meatBefore treatment, the average pH of the minced meatsamples was 5.4, but treatment with propionic acidresulted in an immediate decrease in pH (0.41 mol/Lpropionic acid to pH 4.2; 0.136 mol/L to pH 4.8).Combinations of propionic and ascorbic acid decreasedpH to values between 4.2 and 4.8, and these values weremaintained throughout storage. The pH of the water-treated control sample increased gradually duringrefrigerated storage. Similar pH increases have beenreported (14) during refrigerated storage of groundbeef. It was reported that beef samples with a higherpH ( ≥ 5.6) had higher counts of Gram-negative bacte-ria, the most dominant being Pseudomonas spp. (15).Pseudomonads use up the available carbohydrate andthen deplete the proteins, giving low-molecular weightpeptides as well as decarboxylated amino acid deriva-tives. Meat that has a high initial pH has low amounts ofcarbohydrates available, so Pseudomonas start attack-ing proteins immediately, increasing the pH of themeat, so it spoils faster (16).When data for Pseudomonas counts and TACs werecompared, they showed virtually the same profiles ofgrowth, indicating that pseudomonads dominated themicroflora of refrigerated minced meat and could beused as indicators of contamination by spoilage micro-organisms (data not shown). The effect of solutions ofpropionic and ascorbic acid on Pseudomonas counts,for minced pork stored for 13 d at 4°C is shown in Fig.1. Growth of Pseudomonas in untreated minced porksamples increased exponentially during refrigeratedstorage, with no lag period, whereas growth on thetreated meat samples was significantly reduced orprevented. No growth occurred in any treated samplesin the first 8 d. After 8 d refrigerated storage,Pseudomonas counts increased for minced pork treatedwith 0.136 mol/L and 0.41 mol/L propionic acid,showing a 1 and 3 log increase, respectively, whereasother acid-treated samples still showed an extended lagphase. In the water-treated control samples, the pseu-

domonads reached the critical spoilage level of log10

7–8 cfu/g for fresh meats (17), after about 13 d ofstorage at 4°C. However in acid-treated meat samples,pseudomonad counts were kept well below the criticalspoilage level and never reached > 105 cfu/g. Theprincipal effect of acidification on pseudomonadsappears to be a lengthening of the lag phase. Overall,0.41 mol/L propionic acid appeared to exert thegreatest antimicrobial effect, having an initial bacteri-cidal followed by a bacteriostatic effect. Combinationsof propionic and ascorbic acids and 0.136 mol/Lpropionic acid alone appeared to have a bacteriostaticeffect only. Ascorbic acid is the stronger acid, with apKa value of 4.1 (18) compared with the pKa value of4.87 (18) for propionic acid. However, ascorbic acid isrelatively unstable and easily broken down.It is generally accepted that it is the lipophilic,undissociated acid molecule that is responsible for thepreservation effect of organic acids, as unchargedmolecular units generally penetrate cells more readilythan do electrically charged ions (19).Antimicrobial effects of ascorbic acid in the presence ofmetal ions have been reported; these were suggested toresult from the generation of hydroxyl radicals, whichthen attack biological molecules (20). An alternativeexplanation for bacterial growth inhibition is thatdehydroascorbic acid, the product of ascorbic acidoxidation during radical formation, causes growthinhibition (21).These marked reductions in numbers of pseudomonadsare promising from the standpoint of food spoilage. The

Fig. 1 Pseudomonas growth during refrigerated storage (4 °C)of minced pork, treated with water (control), or solutions ofpropionic acid alone or in combination with ascorbic acid.Each data point is the average of two determinations.Variation was typically ± 3.3% of the average. (–r–) control;(··j··) 0.136 mol/L propionic acid; (··m··) 0.41 mol/L propionicacid; (–e–) 0.136 mol/L propionic acid, 0.057 mol/L ascorbicacid; (–h–) 0.102 mol/L propionic acid, 0.014 mol/L ascorbicacid; (–s–) 0.204 mol/L propionic acid, 0.028 mol/L ascorbicacid; (–n–) 0.306 mol/L propionic acid, 0.043 mol/L ascorbicacid

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results of the present study indicate that acid treatmentincreases the shelf-life of meat (with respect to micro-bial quality) by > 7 d at 4 ± 1°C, assuming that 7 log10

cfu/g meat is the acceptable limit of microbial quality.

Effects of pH on the texture of minced porkDecreases in pH for all acid-treated meat samplescoincided with greater volumes of exudate, comparedwith water-treated control samples. Increase in exudatewas measured as the WHC/g dry weight of mincedmeat. WHC can be defined as the ability of meat tohold its own or added water when subjected tomechanical forces. Within the acid treatments con-sidered, 0.41 mol/L propionic acid had the lowest pHand WHC. Minced meat treated with 0.136 mol/Lpropionic acid and combinations of propionic (0.136mol/L) and ascorbic (0.057 mol/L) acids, showed thegreatest retention of water (data not shown). Previouswork (22) has suggested that shrinkage of meatmyofibrils due to a lower intracellular pH caused driploss by reducing the spaces between the three-dimen-sional network of myofibrils that are able to retainwater.

Effect of pH on the colour of the meat surfaceAll acid-treated samples had higher Hunter L valuesthan did water-treated controls, indicating a bleachingeffect on the surface of the minced meat. A decrease inpH caused fading of meat colour due to denaturation ofthe globin protein moiety that protects the heme (23).Acids are also known to cause oxidation of myoglobinto metmyoglobin. Minced pork samples treated with0.41 mol/L propionic acid were significantly lighter andless red (higher Hunter L and hue values, respectively)than minced pork from other acid treatments. Figure 2shows the effect treatments had on hue values; alltreatments gave similar degrees of variation betweenreplicates, so only error bars for samples treated withpropionic acids and ascorbic acid alone and the controlare shown. From Fig. 2, it can be seen that treatmentscontaining a combination of propionic and ascorbicacids resulted in lower hue scores (more red) than didtreatment with 0.41 mol/L propionic acid alone. Using alower concentration of propionic acid or a combinationof ascorbic acid and propionic acid appears to have aprotective effect on meat colour (24), which has beenattributed to lower metmyoglobin formation. Variousaccounts of the chemistry of meat pigments and theinterrelationship between the different forms havebeen published (25–27). Many of the reactions ofpigments involve oxidation or reduction. Ascorbic acidis able to reduce or delay metmyoglobin formation as itis an effective reducing agent, therefore helping tomaintain the natural meat colour, which is of consider-able importance when selling meat. However, ascorbicacid can only preserve the existing colour of the meat;it cannot enhance or augment meat colour. Chromavalues were not affected by storage time, nor by acidtreatments.

Odour of acid-treated meat samplesA major factor influencing the quality of foods, andespecially meat products, is lipid oxidation, whichresults in rancid odours. The susceptibility of musclefoods to oxidative changes has been related to theproportion and degree of unsaturation of fatty acids intheir lipids, as well as the presence of proxidant andantioxidant factors and oxygen. The oxidative changesin meat products occur through autocatalytic-typereactions, which produce several byproducts that con-tribute to the rancid odours and flavours.The actual amounts of volatile found in replicates of thesame treatment and storage time showed some varia-tion, and thus an external standard was used to takeinto account variation in peak areas of volatiles due toinstrumental drift. Variation was typically 24%, whichshould be taken into account when interpreting thedata in Figs 3 to 7. The general patterns of volatileproduction for some of the key volatiles present in theminced pork samples (Figs 3 to 7) show a number offurans, alcohols, sulphides, aldehydes and ketones.Most of the compounds identified were carbonyls,which arise principally from lipids. It has been found(28) that total carbonyl concentration in L.dorsi dou-bled after 3 d storage, and monocarbonyl contentincreased threefold. This pattern of carbonyl produc-tion was observed in the water-treated control samplesstored over 13 d at 4°C. The effect of individualcompounds on the sensory quality of meat dependsupon their concentration and odour thresholds. Thealdehydes are particularly important in this respect due

Fig. 2 Hue values for minced pork stored (4 °C), treated withwater (control), or solutions of propionic acid alone or incombination with ascorbic acid. Each data point is the meanof three determinations. Error bars show typical variation( ± sx) for these measurements. (–r–) control; (··j··) 0.136mol/L propionic acid; (··m··) 0.41 mol/L propionic acid; (··*··)0.057 mol/L ascorbic acid; (–e–) 0.136 mol/L propionic acid,0.057 mol/L ascorbic acid; (–n–) 0.306 mol/L propionic acid,0.043 mol/L ascorbic acid; (–s–) 0.204 mol/L propionic acid,0.028 mol/L ascorbic acid; (–h–) 0.102 mol/L propionic acid,0.014 mol/L ascorbic acid

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to their low odour thresholds, making them sensoriallysignificant. It can be seen from Fig. 3 that hexanal wasthe most abundant aldehyde produced in the propionicacid-treated samples. The volatile has a ‘green odour’and is formed from the oxidation of linoleic acid.

2-Pentyl furan (Fig. 4) and 1-octen-3-ol (Fig. 5) areother products of lipid oxidation. 2-Pentyl furan isreported to be formed from the conjugated dieneradical generated from the cleavage of the 9-hydroxyradical of linoleic acid. This diene radical may reactwith oxygen to produce a vinyl hydroperoxide that willundergo cyclization via the alkoxy radical to yield2-pentyl furan (29). The same pattern of production wasobserved for 2-ethyl furan (Fig. 6) and 2-pentyl furan.1-Octen-3-ol has a mushroom odour; in meat, it couldbe formed from the 13-hydroperoxide linoleic acid.It can be observed from these data (Figs 3 to 6) thattreatment of minced raw pork with combinations of

Fig. 3 A comparison of hexanal production during refriger-ated storage (4°C) of minced pork, treated with water(control), or solutions of propionic acid alone or in combina-tion with ascorbic acid. Each data point is the average of twodeterminations. Variation was typically ± 18% of the average.(–r–) control; (··j··) 0.136 mol/L propionic acid; (··m··) 0.41mol/L propionic acid; (–e–) 0.136 mol/L propionic acid, 0.057mol/L ascorbic acid; (–h–) 0.102 mol/L propionic acid, 0.014mol/L ascorbic acid; (–s–) 0.204 mol/L propionic acid, 0.028mol/L ascorbic acid; (–n–) 0.306 mol/L propionic acid, 0.043mol/L ascorbic acid

Fig. 4 A comparison of 2-pentyl furan production duringrefrigerated storage (4 °C) of minced pork, treated with water(control), or solutions of propionic acid alone or in combina-tion with ascorbic acid. Each data point is the average of twodeterminations. Variation was typically ± 10% of the average.See Fig. 3 for key to symbols

Fig. 5 A comparison of 1-octen-3-ol production duringrefrigerated storage (4 °C) of minced pork, treated with water(control) or solutions of propionic acid alone or in combina-tion with ascorbic acid. Each data point is the average of twodeterminations. Variation was typically ± 32% of average. SeeFig. 3 for key to symbols

Fig. 6 A comparison of 2-ethyl furan production duringrefrigerated storage (4 °C) of minced pork, treated with water(control), or solutions of propionic acid alone or in combina-tion with ascorbic acid. Each data point is the average of twodeterminations. Variation was typically ± 22% of the average.See Fig. 3 for key to symbols

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propionic and ascorbic acid reduced lipid oxidationvolatiles compared with propionic acid treatment alone.However, most ketones and 3-methyl butanal, whosepattern of production is shown in Fig. 7, did not behavein this manner. 3-Methyl butanal has a malty greenodour and is the Strecker aldehyde derived fromisoleucine. It has been reported as one of the major endproducts of the metabolism of Brochothrix thermos-phacta and possibly Pseudomonas species. Oxidativedeamination of the amino acid and decarboxylation ofthe corresponding α-keto acid is carried out to obtainenergy from amino acids without the use of a hydrogenacceptor.Previous work (11) has shown that treatment of mincedmeat with either lactic or propionic acids increasedlipid oxidation volatiles, and the amount of volatileincreased with increasing concentration of acid andstorage time. The pH value of 6.1 has been identified asthe point above which lipid oxidation is retarded, whileat lower pH values, it is enhanced. This phenomenonhas been attributed to the ionization of histidineresidues at pH values lower than 6.1, which may releasetrace metal catalysts and enhance oxidation.The low production of lipid oxidation volatiles noted inFigs 3 to 6 in minced pork treated with combinations ofpropionic and ascorbic acids is probably due to theprotection of lipids by the antioxidant ascorbic acid, asit is preferentially oxidized in place of other substrates.The antioxidant properties of ascorbic acid are due tothe enediol grouping. In aqueous solutions, particularlyin a neutral or alkaline medium, ascorbic acid willreadily decompose but in a slightly acid medium it ismore stable.Ascorbic acid can act as a primary antioxidant undersome conditions by absorbing free oxygen in a closed

system. Ascorbic acid used in conjunction with otherantioxidants has been observed to be effective inpreventing oxidative changes. It probably acts bydonating its hydrogen and regenerating natural oradded antioxidants.

Conclusion

Combinations of ascorbic and propionic acid solutionswere used in an effort to reduce the formation of lipidoxidation volatiles formed on pork mince meat treatedwith solutions of lactic and propionic acids (11). It canbe concluded that, by using a treatment of ascorbic acidon its own or in combination with other organic acidson the surface of meat, it is possible to control thegrowth of potent spoilage bacteria and thereforeincrease the shelf-life of the meat, while minimizing theproduction of off-flavours derived from lipidoxidation.Significant reductions in TAC and Pseudomonas countswere noted for all concentrations of acids used,increasing the shelf-life of the meat at 4°C to more than7 d, compared with 5 d for a water-treated controlsample.Ascorbic acid in combination with propionic acidinhibited pigment oxidation, maintaining redness at themeat surface. Low hue values are associated with freshmeat products, indicating a uniform and stable redcolour. In comparison, a propionic acid-treated samplesuffered bleaching (high hue value) on the surface ofthe meat, particularly at higher concentrations of acid,which are necessary to extend the shelf-life of themeat.There appears to be a direct link between 3-methylbutanal production and possible pseudomonad and B.thermosphacta growth (cf. Figs 1 and 7). This isimportant as 3-methyl butanal production, which isproduced from the decomposition of the amino acidisoleucine, is responsible for a green malty odour. Oncebacteria start to attack proteins, the pH of the meat willrise due to breakdown products such as ammonia beingproduced, increasing the rate of meat spoilage. By usingan acid decontamination, it is possible to significantlyreduce the growth of these bacteria and thus eliminatethe production of this volatile, thereby increasing theshelf-life of the minced meat.These results show the benefits of using ascorbic acid incombinations with propionic acid as it prevents loss ofmeat colour, reduces the amounts of off-odours asso-ciated with lipid oxidation and improves the microbio-logical quality of the meat during storage at 4°C.

Acknowledgement

This work was funded by a grant from the EuropeanUnion under the International Scientific Co-operationInitiative.

Fig. 7 A comparison of 3-methyl butanal production duringrefrigerated storage (4 °C) of minced pork, treated with water(control), or solutions of propionic acid alone or in combina-tion with ascorbic acid. Each data point is the average of twodeterminations. Variation was typically ± 39% of the average.See Fig. 3 for key to symbols

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