membrane filtration offoodsuspensions · of membrane filter, flow direction, pressure differential,...

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1979, p. 21-350099-2240/79/01-0021/15$02.00/O

Vol. 37, No. 1

Membrane Filtration of Food SuspensionsANTHONY N. SHARPE,* PEARL I. PETERKIN, AND ISTVAN DUDAS

Bureau ofMicrobial Hazards, Food Directorate, Health Protection Branch, Health and Welfare, Ottawa,Ontario KIA OL2, Canada

Received for publication 23 October 1978

Factors affecting the membrane filtration of food suspensions were studied for58 foods and 13 membrane filters. Lot number within a brand, pore size (0.45 or0.8 pm), and time elapsed before filtration had little effect on filterability. Brandof membrane filter, flow direction, pressure differential, age (microbiologicalquality) of the food, duration of the blending process, temperature, and concen-tration of food in the suspension had significant and often predictable effects.Preparation of suspensions by Stomacher (relative to rotary blender) addition ofsurfactant (particularly at elevated temperature) and prior incubation with pro-teases sometimes had dramatic effects of filterability. In contrast to popularopinion, foods can be membrane filtered in quantities pertinent to the maximumsused in conventional plating procedures. Removal of growth inhibitors and fooddebris is possible by using membrane filters. Lowering of the limits of detectionof microorganisms by concentration on membrane filters can be consideredfeasible for many foods. The data are particularly relevant to the use of hydro-phobic grid-membrane ifiters (which are capable of enumerating up to 9 x 10'organisms per filter) in instrumented methods of food microbiological analysis.

Sharpe and co-workers (22, 23, 25-27) showedthat a novel growth vehicle-the hydrophobicgrid-membrane filter (HGMF)-has great at-tractions in the automation of enumerative mi-crobiology because of its potential ability tosimplify the engineering requirements for dilut-ing and counting and to eliminate the likelihoodof false low counts at high levels of contamina-tion. Its attractions also extend to manual count-ing procedures, because its use could often elim-inate the need to make dilutions and the loss ofdata through misjudging contamination levels.Demonstration of the ability of HGMF to

provide linear recoveries at levels as high as 9x 10' colony-forming units per filter (26) andmaintain a linear performance with suspensionsof several common foods (23) reinforced hopesthat the unique properties of the HGMF mightbe exploited in the instrumentation of generalfood microbiology. However, thorough knowl-edge of the filterability of food suspensionsthrough membrane filter (MF) material and thefactors affecting it was seen to be an importantrequirement before further developments inHGMF-based instrumentation could be consid-ered. A literature search revealed an apparentapathy towards MF techniques by food micro-biologists; little data on the membrane filtrationof foods, at levels equivalent to those used in,for example, plate count analyses, appear toexist.

The most relevant published uses of MF arerestricted to easily filterable beverages such aswines, beers, and sugar liquids (10-12, 15, 19, 20)and to a few dairy products such as milk, butter,and ice cream (1-4, 6, 8, 9, 16-18). The mem-brane filtration of wines, beers, and sugar liquidsfor microbiological analysis would appear topresent little problem. An interesting method ofimproving the filterability of egg albumin forSalmoneUa determinations by pectic enzymehydrolysis followed by clarification with celitewas described by Kirkham and Hartman (14).Apart from this, the published uses of MF infood microbiology appear to be limited to filtra-tion of swabbed or shaken extracts (7) and apressure method of transferring surface orga-nisms to the MF (28). The American PublicHealth Association Compendium (29) recom-mends microbiological analysis by filtration forfoods which "can be dissolved and passedthrough a bacteriological membrane filter....,"without, however, offering any examples of suit-able subjects.There has been more enthusiasm from the

dairy industry, but the problems and solutionshave not been well defined. Some workers reportdifficulty filtering milk, for example (8, 17). Theproblem was avoided in one case by centrifugingand resuspending the milk (4). Improvement infilterability resulting from dilution of the speci-men has been claimed (9, 18). A rather uncon-

21

on January 12, 2021 by guesthttp://aem

.asm.org/

Dow

nloaded from

http://aem.asm.org/

22 SHARPE, PETERKIN, AND DUDAS

trolled method, whereby a milk sample to befiltered was added to warm (45 to 5000) surfac-tant solution already in the filter, was describedby Fifield and co-workers (6) and others (2, 3).In another case, ice cream was made filterableby liquefaction, settling, and addition of surfac-tant (16).Apart from these examples, the microbiologi-

cal applications of MF appear to be limited tothe analysis of waters (which needs no furthercomment) and the sterilization of fluids. Theabsence of publications pertinent to foods wasnot surprising because (i) the numerical opera-ting range of the ubiquitous 47-mm MF is sosmall (e.g., lower and upper counting limits of20 and 80 colonies, respectively) that many diskswould be required per analysis to accommodatethe range of counts found in most foods, and (ii)most microbiologists in our experience tend todismiss food suspensions as being generally un-filterable.The HGMF has a numerical operating range

far greater than any conventional MF (or evenpetri dish) so that point (i) above does not nowapply. The potential applicability of MF meth-ods to food microbiology thus rests on the valid-ity of point (ii). There seemed to be so muchunverified opinion about the factors affectingmembrane filterability that we decided to obtaina definitive data base for any future work oneither HGMF or MF.

MATERIALS AND METHODSFiltration apparatus. The filtration apparatus is

shown in Fig. 1. Positive pressure was used above theMF rather than the normal underside vacuum, toallow more precise control of pressure differential. No

APPL. ENVIRON. MICROBIOL.

significant alteration in flow rate was expected, except,perhaps, where boiling or drastic outgassing of thefiltrate may occur under excessive vacuum. MF sup-port was provided by a flattened stainless steel meshA (Michigan Wire Cloth, Ambac Industries, GardenCity, Mich.; 40 by 40; 0.010-inch [ca. 0.025-cm] wire).The extent to which this may have interfered withflow rates was not calculable, but is believed to benegligible. Cylinder B contained the sample, and de-fined the filtration area (1.905-cm diameter, 2.850 cm2).The apparatus was capped and sealed with screwclamp (C). Pressure, applied through a valve D via atwo-stage reducer from a nitrogen cylinder and moni-tored by an open mercury manometer, varied by lessthan 1% during a filtration. The filtrate outlet heightwas arranged to be level with disk A; a small, addi-tional time-variable pressure resulting from the headof sample in B was ignored. Valve E allowed filtrate tobe piped to a Sartorius 3716MP electronic balance(Canadian Laboratory Supplies Ltd.) fitted with adraft-excluding cover and a suction line for removal offiltrates. Analog output from the balance was recordedon tracing paper by a Servogor recorder (Brinkmann2541, Brinkmann Instruments, Canada Ltd.) runningat 60 mm/min.MF. In all cases, the manufacturer's orientation of

MF material was preserved. The following MF werepurchased as 25-mm disks or were punched from largerformat material already on hand: Amicon microporousfilter, 0.45 ,um (Amicon Canada Ltd.), lot number BB0050F SUBA; Gelman Metricel, 0.45 ,um, 6N, lot num-ber 81913, and GA-6, lot number 81859; Gelman Me-tricel 0.8 ,um, 4N, lot number 81264, and GA-4, lotnumber 81681 (Gelman Instrument Co.); Millipore0.45 pm HAWP, lot numbers 22819, 14247-19, 109876,106515, and 26945-19, 0.45 Am HABP, lot number09242-3; 0.8 jum AAWP, lot number C7M21886A (allMillipore Ltd., Mississauga, Ont., Canada) and Mill-pore 0.45 ,um HABP, lot number unknown (BDHChemicals Ltd.); Oxoid Nuflow 0.45 gim, lot number3722, and 0.8 ,um, lot number 3680 (Medex Chemicals

FIG. 1. The filtration apparatus with (a) and without (b) cylinderB inplace. For explanation ofcomponents,see text.


.asm.org/

Dow

nloaded from

http://aem.asm.org/

MEMBRANE FILTRATION EFFECTS 23

Ltd.); Sartorius 0.45 inm SM11306, lot number061233726, and 0.45 jam SM11106, lot number060964702; Sartorius 0.8 um SM11304, lot number300814572, and SM11104, lot number 401836572 (BDHChemicals Ltd.). Unless otherwise stated, all data referto filtration through Millipore HAWP material, lotnumber 106515.

Foods. The following foods were purchased locallyand stored at ambient, 4 or -20°C as appropriate:bacon; beef chunks; lean (15.6% fat), medium (25.0%fat), and regular (30.1% fat) ground beefs; frozenchicken; frankfurters (Maple Leaf); ground pork(31.2% fat); pork sausages (Maple Leaf); canned clams(Clover Leaf); cod fish sticks (High Liner); oceanperch; cooked shrimp (Brilliant); turbot; apple sauce(Stokely); baked beans (Heinz); frozen brussels sproutsand frozen carrots (Arctic Gardens); pickled gherkins(Bick); frozen green beans (Loblaw); lima beans(York); canned mushrooms (Clover Leaf); potato(Dollar Chips, McCain); frozen strawberries (York);basil, ground cinammon, and ground nutmeg (allMcCormick); walnuts (McNair); whole and skim milk(Clark); skim milk powder (Carnation); table cream(Borden); sour cream (Sealtest); vanilla ice cream(Loblaw); yoghurt-Set Style and Swiss Spun (bothDelisle); butter (Loblaw); cheddar cheese (Black Dia-mond); mozarella, Philadelphia, and Velveeta cheeses(all Kraft); apple turnovers (Pepperidge Farm); break-fast cereal (Quaker's Captain Crunch); Egg Beaters(Fleischmann); vanilla instant pudding (Jello); pizza(Gusto); puff pastry (Gainsborough); ravioli with meat(Nelia); spaghetti (Primo); turkey pot pie (Loblaw);frozen waffles (Loblaw); chicken and rice instant soupmix (Loney); vegetable beef soup (Aylmer); tomatosoup (Campbell); mushroom soup (Clark); apple juice(Martin); and orange juice (Loblaw). For age studies,frozen or refrigerated subsamples were removed to thebench (room temperature 22 ± 2°C) at appropriateintervals before the experiment.

Filtration and recording. The operational detailsshould be self-evident, and only the following pointsare noted. For each new filter disk, a preliminary runwas made with distilled/deionized water to clear anybubbles from the line. With the unit pressurized (valveD) and the recorder zeroed, with the chart in motion,valve E was opened to pass filtrate when the chart wasat the desired (x-coordinate) position. In this way,several corresponding curves could be drawn from thesame starting point to aid visualization. Incompletelyfiltered samples were aspirated out before removingB. The data are displayed in terms of weight of foodfiltered out per square centimeter of MF (rather thanvolume of suspension) unless otherwise indicated.

Preparation of food suspensions. Decimally di-luted suspensions in distilled/deionized water wereprepared from 10- to 40-g specimens according to thegeneral homogeneity of the food. Suspensions wereprepared in Mason jars on an Osterizer blender (Sun-beam Corp. of Canada Ltd.) at 25,000 rpm, or in aStomacher 400 (Canadian Laboratory Supplies Ltd.,or Dynatech Corp.). Centimally diluted suspensionswere prepared separately by blending 4 g of food in396 ml of diluent. For experiments on time elapsedbefore filtration, suspensions were transferred to beak-ers and either stirred continuously (magnetic stirrer)

at room temperature or allowed to settle and stirredimmediately before use. Except where otherwisestated, blending times were 60 s for the Osterizer and30 s for the Stomacher, suspensions were used within15 min of preparation.Pressure differential studies. Headspace pres-

sures of 10, 20, 30, 60, and 95 kPa (101.4 kPa = 760mm of Hg) were employed. The highest figure isequivalent to the pressure drop achieved by a goodwater pump. Investigation of higher pressures wasdeemed unnecessary. Unless otherwise stated, datarefer to a pressure differential of 95 kPa.

Surfactant studies. In preliminary studies, foodswere blended in Triton X-100 and Tween 80 solutions,up to 10% (wt/vol). Most of the data reported hereare for 1% solutions of these surfactants. For stomach-ing, air was removed from the bag by sliding it downinto the stomaching compartment; this avoided thefrothing which is inevitable with the rotary blender.

Protease studies. Trypsin (Nutritional Biochemi-cal Corp., Cleveland, Ohio) at a final concentration of100 jig/ml in 0.1 M tris(hydroxymethyl)aminometh-ane-hydrochloride buffer, pH 7.8, was stomached withfood (10%) and incubated for up to 70 min at 37°Cbefore filtration. Similar suspensions with Pronase(Calbiochem, Los Angeles, Calif.) at the same concen-tration in 0.02 M sodium acetate buffer, pH 6.0, werealso incubated at 37°C for up to 70 min.Temperature dependency studies. The filtration

unit and suspensions were immersed in a water bathand equilibrated. The unit was raised from the bath toadd MF, water, or suspension, and returned immedi-ately. Where not stated, data refer to ambient tem-perature (22 ± 20C).

RESULTSGeneral considerations regarding filter-

ability. The filterability of a suspension of foodin water, under a constant pressure differential,may be interpreted in several ways. Filtrationrate (the slopes of the graphs) defined as eitherthe volume of filtrate issuing from the filter orthe weight of food brought into intimate contactwith the filter (the cake) per second, is relativelymeaningless, changing as it does while filtrationprogresses. At the instant of commencing filtra-tion, every food suspension had a high filtrationrate (Q0) indistinguishable from that of water.Thereafter, the rate decreased as a function offood, preparation conditions, temperature, etc.In some instances filters became quite imperme-able when a certain amount of food had beenfiltered, whereas in others a more or less steadystate was reached. A cake filtration model (13),which assumes that flow rate decreases in in-verse proportion to the amount of food accu-mulated on the filter, leads to the following flowrate equation:

t - aiv + a2t (1)

(v = volume, t = time). This relation, which

VOL. 37, 1979


.asm.org/

Dow

nloaded from

http://aem.asm.org/


would have allowed comparisons to be madeeasily from tables of the constants a, and a2, wasquickly shown to be inadequate at describingthe shapes of many of the graphs. Some curvesare better described by a complete blockingmechanism (13) in which particles plug porescompletely, i.e., by the following relation:

v = Qo(1 - e-kt) (2)

Brief examination of the data by using thefollowing general model:

t= aO + alv + a2V2+ a3V3+... (3)

led to sets of figures from which the practicalitywas only discernible by recalculating the curves.Other forms of data, for example, tables of quan-tity filtered during various time intervals, be-came cumbersome when attempting to comparethe effects of different variables. It was decided,therefore, that the curves themselves conveyedinformation in the most practical and compactmanner.

Intrinsic variability of filtration rates.Figure 2 exemplifies the many graphs obtained

HlILLIPOREALL. L

fSomached

0.1

0.10

by filtering stock food suspensions through dif-ferent manufacturer lot numbers and differentblendings of the same food specimen throughseveral MF from one lot number. Successivefilterings of stock suspensions through MF froma given lot number yielded almost perfectly su-perimposable graphs, and the effect is not illus-trated in Fig. 2. However, successive blendingsof the same food specimen yielded minor varia-tions which could either be due to inhomogene-ity of the specimen or blending inconsistencies.Similarly, there were differences between lotnumbers from the same manufacturer; Milliporelot number 22819, for example, consistently fil-tering slower than lot number 106515.Effect of filter brand and pore size. Fig-

ures 3 and 4 illustrate that for ground beef, greenbeans, turbot, and milk, at least, permeabilitydifferences between the MF brands in a givenpore size were as great as or greater than thosebetween pore sizes in a given brand. In fact,differences in flow rate between 0.45- and 0.8-,umpore sizes of a brand were generally barely no-ticeable, and for stomached ground beef theOxoid Nuflow 0.8-,um MF actually filtered less

E HAWPOTS

a potato

f LOT 106515

Stomaohed< / ground beef

LOT 22819

_~ LOT 106515Blended potato

LOT 22819

TIME - EACH DIVISION = 1o SEC

FIG. 2. Variation ofmembrane filtration rate with lot number in Millipore MF. The three curves for eachlot (center and right hand groups) are different blendings of the same sample.



.asm.org/

Dow

nloaded from

http://aem.asm.org/

MEMBRANE FILTRATION EFFECTS

a

04

49B9

Ground beef

25VOL. 37, 1979

4N

0

go

0

I..

0N

a*R -

go

rA

34

Green beaus /

aop

z

Turbot

00SE

49

e eO0 aS

-

S

0

S9

a0k

a

TIME -EACH DIVISION = 10 SEC

FIG. 3. Variation ofmembrane filtration rate with MF brand andpore size, for stomached (S) and rotary-blended (B) ground beef, green beans, and turbot.


.asm.org/

Dow

nloaded from

http://aem.asm.org/


rapidly than the 0.45-,um pore size. The differ-ences between filters for these foods do notcorrespond to the relative flow rates determinedfor water, shown in Table 1.

Effect of flow direction through MF. Fig-ure 4 shows that flow direction had a very pro-nounced effect for all the 0.45-pn filters, exceptthe Sartorius cellulose nitrate with milk. Allfilters except both the Sartorius (cellulose ace-tate and nitrate) were more permeable whenused in the orientation supplied (i.e., with thesuspension to be filtered in contact with the sideuppermost, as packed). These findings should becompared with the following manufacturer's in-structions regarding orientation: Amicon, "In-sert either side up"; Gelman, "Facing up towardsmaterial being filtered"; Millipore, no instruc-tions; Oxoid, "Filter through top as packed";Sartorius, no instructions. The cause of the var-iability is undoubtedly complex; the large differ-ence observed for Millipore filters with milk doesnot follow the small differences for stomachedground beef, green beans, and turbot, for exam-ple.Effect of pressure differential. The filtra-

tion rate of water (and presumably of food sus-pensions also, at the instant of commencing fil-tration) varied with the type of MF, but wasalways proportional to the pressure differentialacross the MF. The coefficient was, for example,0.009 g/cm2 per s/kPa for the 0.45-,um-pore-sizeSartorius cellulose acetate MF (Table 1). Withfoods, however, although increasing pressure dif-ferential always resulted in faster filtration, theeffect rapidly became nonlinear; after 10 or 60 s,for example, the quantity filtered at 95 kPa wasalways less than 9.5 times that at 10 kPa (Fig.

TABLE 1. Flow rates and specific flow rates ofwater through MF at 20°C

Flow at 95 SpecificFilter Pore size kPa flow rateFilter (AM) (W/m2 (W/m

per s) pers/kPa)Amicon 0.45 1.18 0.0124Gelnan GA-6 0.45 1.10 0.0115Gelman GA-4 0.8 1.90 0.0201Gelman 6N 0.45 1.14 0.0120Gelman 4N 0.8 2.00 0.0211Millipore HAWP (lot 0.45 0.83 0.0087

no. 106515)Millipore AAWP 0.8 2.22 0.0234Oxoid Nuflow 0.45 1.00 0.0105Oxoid Nuflow 0.8 1.60 0.0168Sartorius SM11306 0.45 0.85 0.0090Sartorius SM11304 0.8 2.00 0.0211Sartorius SM11106 0.45 1.07 0.0112Sartorius SM11104 0.8 2.00 0.0211

5). The effect of pressure differential becameless important as filtration progressed, but in noinstance was the quantity filtered at a low pres-sure observed to overtake that from a higherpressure.

Effect of time elapsed before filtration.No significant changes were observed with eitherstomached or blended ground beef and potato,when suspensions were either stirred or allowedto stand for up to 45 min before filtration.Graphs were virtually superimposable and arenot shown.

Effect of suspension preparation proce-dure. Except in the case of vanilla ice cream, forwhich the blended specimen filtered more rap-idly than the stomached one, and Egg Beaters,for which there was no noticeable difference, allfood suspensions prepared by Stomacher filteredmore rapidly than those prepared by rotaryblender (see Fig. 6, 7, and 9). The differenceswere dramatic for relatively intact foods (e.g.,carrots and beef chunks) but decreased in im-portance for those foods which were alreadydisrupted or dispersed by the manufacturingprocess (e.g., ground beef, soups). As would beexpected, also, from the continued breakdown oftissues into smaller particles, increased blendingtime in either instrument resulted in decreasedfilterability (Fig. 7).

Effect of food concentration. Although thequantity of filtrate obviously increases with de-creasing concentration offood in the suspension,the total weight of food brought into contactwith the MF may change either way (Fig. 8).Thus, chicken became less, whole milk slightlymore, and skim milk much more filterable.Effect of age of the food. The filterability

of both stomached and blended ground beef,green beans, and turbot decreased with increas-ing age up to 48 h (Fig. 9). This trend might beexpected from the decreasing tissue integritycaused by autolysis and microbial growth andthe increased microparticulate content of thesuspensions which would result. In contrast, thefilterability of (stomached) whole milk increaseddramatically by 48 h-a consequence of the re-moval of particulate material during clotting,which was not effectively resuspended by thehomogenization.Effect of surfactant at 20°C. At this tem-

perature, the effect of adding 1% Tween 80 orTriton X-100 was very variable. The filtrationrate for stomached ocean perch increased mod-erately with Triton X-100 and greatly withTween 80 added (Fig. 10). Whole milk andblended green beans filtered slightly faster withsurfactant added, whereas stomached groundbeef and blended potato filtered less rapidly



.asm.org/

Dow

nloaded from

http://aem.asm.org/

aso

".

.

4

0

go

J

0

AU

MEMBRANE FILTRAT

*6UKtIN *nloau;jot

a 0

U&1-V

ALOIJUN PIOZO0

0) a

JEYR .oid,jti

a

dATH eaodltI,K

9-yo Ulot.o

0

a

it0

e:4

It a

_ :I"0.5.3I'

l* d

an Z4

a

P a

mS uWUIoO

wselmv

o.

is

1a

1~0a.

IDa~~~

OA~~~I.Is

3~g

t)a ~

I

a

a

UEZ IA

ION EFFECTS

I.

ew

'40

',is

w

-is

Z.t

13

co

Vo.. 37, 1979 27

- on January 12, 2021 by guesthttp://aem

.asm.org/

Dow

nloaded from

http://aem.asm.org/


0.

ofa

C)

04

02

04

Sto oh-oedgrouad beef

Stoemehedpotato

10 kP

Whole milk1: IO&

.95

.20

-10

Whole milk1: 100

TIME -EACH DIVISION = 10 SECFIG. 5. Variation ofmembrane filtration rate with pressure differential, a, Vertical scale correct; b, divide

vertical scale by 10.

(Fig. 10). Vegetable beef soup and Velveeta (Fig.11) were not noticeably affected by addition ofTriton X-100. Its addition to stomached butter(Fig. 11) decreased the filtration rate dramati-cally, and higher concentrations (e.g., 2, 5, and10%; data not shown) decreased the rate stillfurther.Effect oftemperature without surfactant.

Over the range 10 to 400C and without addedsurfactant, increasing temperature produced anincreased filtration rate. For blended potato,vegetable beef soup, Velveeta, and stomachedwhole milk (Fig. 11), the increase was modest.In the first three, the increase is believed to bemainly due to the decreasing viscosity of water.(Thus, plotting the quantity of blended potatofiltered after 15 or 30 s against the reciprocal ofthe viscosity of water at the different tempera-tures yielded an almost straight line; graph notshown). Whole milk ifitered faster at 40°C thanwould be expected from viscosity decrease alone,

indicating that some conformational change maybe important. Stomached butter, which filteredvery rapidly at 20 to 400C, was dramaticallyslower at 10°C-presumably an effect of thecohesivity of fat in the Stomacher bag.Combined effects of surfactant and tem-

perature. The effect of these two factors to-gether was complex. All foods filtered more rap-idly at the higher temperatures, with or withoutTriton X-100. Blended potato filtered less rap-idly at all temperatures with the surfactant pres-ent (Fig. 11), and vegetable beef soup was notgreatly affected (Fig. 11), whereas the poor fil-terability of Velveeta improved very slightly at400C with the surfactant present (Fig. 11). How-ever, concentrations of Triton X-100 up to 5% at400C did not improve the filterability of Vel-veeta further (Fig. 12). Filtration of stomachedbutter at 20 to 400C was dramatically inhibitedby Triton X-100 (Fig. 11); in this case, the sur-factant presumably increased the amount of fat

FIG. 6. Membrane filtration rates for 10% suspensions of 54 common foods. Except for apple and orangejuices, graphs show both stomached (S) and rotary-blended (B) preparations. For data on whole and skimmilk, butter, and ocean perch, see Fig. 5 and 8 to 11.



.asm.org/

Dow

nloaded from

http://aem.asm.org/

*

0. v-~~Sa

04~~~~

m d~~h

A Ah d

h * *0 0

* * .!A i a.* I

0~~~~~~C~ ~ ~wO

TIMZ - ZACK DIVISION u 10 SZC

29


.asm.org/

Dow

nloaded from

http://aem.asm.org/


0.15

atN

N

02NA14

0

0.10.

0.05'

15,30sosee

ItomeebodPotato

15

30/sol

fot.iaeh.dgroundbeef

Blendedpotato

Is3ee30e00

60t630so

Dendedgroundbeef


FIG. 7. Variation ofmembrane filtration rate with suspension preparation time.

Blendedoeieken

1:10

_ 1:100

Whole milk

1:100,'{ 110

Skim milk

1:10

1:1

TIME -EACH DIVISION* 10 SEC

FIG. 8. Variation of membrane filtration rate with food concentration. System sensitivity adjusted tocompensate for decreased food concentration from 1:10 to 1:100 suspensions.

dispersed as globules by the Stomacher. Wholemilk, on the other hand, although being rela-tively unaffected at 10 and 20°C, filtered some-what better at 30°C and dramatically better at35 and 400C (Fig. 11); in this case, fat globuleswere presumably removed by solubilization inthe surfactant.Effect of protease. The dramatic improve-

ment in filterability of Velveeta after 70 min ofincubation with Pronase contrasts markedly

with the insignificant improvement caused byup to 5% Triton X-100 (Fig. 12). Trypsin pro-duced a smaller improvement during this time;the effect of both proteases increased greatlybetween 60 and 70 min, evidence that the distri-bution of particle sizes passes through a criticalrange. Similar improvements were noted forskim milk powder (Fig. 12). In this case, how-ever, the relative effect of the two proteases wasreversed.

a0

as0.05.

0.0s

02

Po

M 4

No4

04



.asm.org/

Dow

nloaded from

http://aem.asm.org/


U

Al

"0 ~ ~~~~X

.A

X

A5

VA

A ~ ~ ~ ~ W

I~~~~~~~so

e

*t

Oh~~~

O~~W *

aWo Uzcd Ui1ISIrId SWVUO

VOL. 37, 1979


.asm.org/

Dow

nloaded from

http://aem.asm.org/


]| A TWEEN

rc~~~~~0

Stomachedground beeta

0.05 Stomached TRITONooean perch TWEEN

Blended Whole milkTITON green beanTRITON TRITONTWEEN TRITON

Blended 0 0IeW Epotato


FIG. 10. Variation ofmembrane filtration rate with addition of 1% surfactant at 20°C. a, Different regularground beef specimen to those in Fig. 2 to 9.

DISCUSSION

The microbiological attractions of MF aregenerally seen to be in situations exploiting theirability to concentrate organisms from a rela-tively dilute and easily filterable specimen, i.e.,lower the limit of detection of the standardplating procedure (29) or allow removal of mi-crobial growth inhibitors (2, 23). There are cer-tainly instances where these properties would bevaluable in food microbiology, for example, inthe determination of Salmonella or yeasts incertain foods. Many of the foods examined inthis study will be seen to be filterable at levelswhich would allow concentration of organisms10-fold or more, compared with conventionalplating procedures. The finding of Sharpe andco-workers (23) that the relative affinities ofcommon food bacteria and food particles forMFare different enough to allow food debris to alsobe selectively removed from the ifiter indicatesthat the MF method has potential for the rapiddetection (or even enumeration) of, say, Sal-

monella. For example, it may be possible toproduce fluorescent antibody-stained coloniesdirectly, having sufficient signal-to-noise ratiofor electronic detection.The membrane filterability of a food suspen-

sion is determined by its microparticulate con-tent. These particles cause a steadily increasingresistance to flow through their adsorption alongthe MF pores or their accumulation as a filtercake. Alternatively, they may completely blockthe pores. At the beginning of this study weexpected that starch or protein particles wouldbe likely to cause the first (standard) type ofblocking and fat or oil globules would cause thesecond (complete blocking). However, the dataindicate that either effect may occur withoutdistinction. Visible particles do not block MFand are preferable because their presence im-plies a lower concentration of smaller particles.The observed effects of suspension preparationby Stomacher and rotary blender are consistentwith those expected from the different mechan-ical and physical actions of the two blending



.asm.org/

Dow

nloaded from

http://aem.asm.org/

MEMBRANE FILTRATION EFFE3CTS

0 0 0a goa a aA A~1

I~* 1

000 0goS0

K00J

0.s

_a.

° '1>

I.~~I

0 000

A~~~

0 0

U. : 2

" \\ \

I

v

i~0 aOa k

o2 2

2: i0 1m I]PO -

M tnm

04 i

I P

rix

04̂ <<

f

,'e

11£-

U . . . .p

a-

0 C--I

ano HadUKUlaliusvuO

VOL. 37, 1979

aa

33

0

° aI_

A40

@0A0J8*** a

; A

L

-9 * * 9

on

m

t

9%

q


.asm.org/

Dow

nloaded from

http://aem.asm.org/


Nx

a

I.4Po

44

so

0

TIME -E ACH DIVISION= 10 SEC

FIG. 12. Relative effects of surfactant (0 to 5% Tri-ton X-100) and protease treatments (70 min) on themembrane filtration rate of Velveeta (left and center).Also relative effects of trypsin and Pronase treat-ments (70 min) on filterability of skim milk powder(right).

methods. The Stomacher is always observed tobe less destructive to tissues than rotaryblenders (5, 21, 24, 31). Use of the Stomacherand the miunimum possible stomaching time foradequate dispersal of organisms are thus to bepreferred for any preparation of samples formembrane filtration.Lot to lot consistency and even choice of 0.45-

versus 0.8-tan pore size from a given manufac-turer appear to be minor considerations in mem-brane filtration. However, the chemical or phys-ical composition ofMF from each manufacturermay be relevant in the reliability of filtration.Similarly, the best direction of flow through theMF should be determined. Direction of filtrationis usually quoted with maximum recovery (par-ticularly of fecal coliform organisms) in mind(30) but its importance to flow rate is obviouslysignificant. Except for the Sartorius filters ex-

amined here, the best direction for filtrationseemed to coincide with the best direction (im-plied) for microbial recovery. However, it shouldbe borne in mind that when the organisms are

filtered in the presence of appreciable quantitiesof food debris any effect on recovery ascribed tosurface roughness, etc., may be considerably per-turbed (most probably being eliminated). In theabsence of further data, filtering in the directionof maximum flow rate is to be preferred.

In the ordinary laboratory filtration set-up,pressure differential is largely uncontrollable,varying according to the pump, ifitration rate,aeration of the sample, etc. There is no evidenceto indicate the desirability of filtering at any-thing other than the maximum pressure differ-ential obtainable with any given apparatus.Warm suspensions are preferable to cold ones,simply on account of the reduced viscosity ofwater.The effect of diluting the sample is most in-

teresting. Reports ofimproved filtration rates ondiluting dairy products (9, 18) are supported byour data. Changes in the degree of dispersal,dissolution and conformation of tissue compo-nents, etc., are obviously important. It is quitepossible that changes in ionic strength, pH, etc.,might also be significant, and these should beinvestigated if difficulties in filtering are experi-enced.

It is apparent that combinations of the use ofelevated temperatures, suitable surfactants, andincubation with proteases (and possibly amy-lases) are capable of producing dramatic in-creases in the filterability of foods, according towhether fat, protein, or starch particles are theprinciple cause of filter pore blocking. Thus, iffiltration difficulties are experienced and if theorganisms are relatively tolerant, an array ofprocedural modifications is available to providethe desired level of filterability. It can be safelyconcluded that most foods are (or can be made)filterable at levels relevant to most microbiolog-ical analytical requirements.From the point of view of the straightforward

trading of many conventional manual agar plat-ing methods (for example, the standard plate,coliform, and Staphylococcus aureus counts) forinstrumented methods based on HGMF, concen-tration of the food is unnecessary. In these cases,it is more important to know whether MF orHGMF of convenient size will filter at least themaximum weight of food encountered in a nor-mal plating procedure within a reasonable timeand particularly what factors limit the proce-dure. For example, ifa procedure is to be carriedout unattended, it is important that the proba-bility of incomplete filtration be very low.The filtration area provided by a typical 6.0-

cm square HGMF of 10,000 grid cells, with hy-drophobic barriers 0.013 cm wide, is 14.4 cm2.Because for most plate counts the greatest quan-tity of food taken is 1.0 ml of a 1:10 homogenate(i.e., 0.1 g) it is required that the filtration curvereliably pass 0.007 g/cm2 within the time allowedfor the food to be considered filterable at a levelpertinent to the conventional analysis. It will beseen from the graphs that, without the use of

ALPPL. ENVIRON. MICROBIOL.


.asm.org/

Dow

nloaded from

http://aem.asm.org/


procedural modifications to facilitate filtration,only Velveeta, Egg Beaters, and skim milk pow-der, out of 58 food specimens, failed to reach thislevel within 15 s, and five others (vanilla instantpudding, Captain Crunch cereal, and vegetablebeef, mushroom, and tomato soups) were in arange where the reliability of filtration might bedoubted. By modifying the preparation proce-dure, even the rather intractable Velveeta couldbe made easily filterable at this level. We con-clude that the attractions ofinstrumented meth-ods of food microbiological analysis based onHGMF need not be limited by the filterabilityof food suspensions and that reduction of thelimit of detection of microorganims from anyfood by concentration using MF is distinctlyfeasible.

ACKNOWLEDGMENISWe are very grateful to M. P. Diotte for tance with

electronic problems, and L. Gray for fat analyses of the meats.

LITERATURE CITED

1. Barber, F. W., C. P. Burke, and H. Fran. 1954. TheMfllipore filter technique in the dairy industry. J. MilkFood TechnoL 17:109-112.

2. Busta, F. F., and AL L Speck. 1965. Enumeration ofBacilus stearothermophilu by use of membrane filtertechniques to eliminate inhibitors present in milk. Appl.Microbiol. 13:1043-1044.

3. Claydon, T. J. 1975. A membrane-filter technique to testfor the significan of sublethally injured bacteria inretail pasteurized milk. J. Milk Food Technol. 8:874-8.

4. Ehrlich, R. 1953. Membrane filter method for determi-nation of coliforms in pasteurized and certified milk. J.Milk Food Technol. 16:6-8.

5. EmBwiler, B. S., C. J. Pierson, and A. W. Kotula.1977. Stomaching vs blending. Food Technol. (Chicago)31:40-42.

6. FMfleld, C. W., J. E. Hoff, and B. K. Proctor. 1957. TheMillipore filter for enumerating coliform orgamams inmilk. J. Dairy Sci. 40:688-689.

7. Frazier, W. C., and D. F. GneIser. 1968. Short-timemembrane filter method for estimation of numbers ofbacteria. J. Milk Food Technol. 31:177-179.

8. Goff, J. H., T. J. Claydon, and J. J. Iandolo. 1972.Revival and subsequent isolation of heat-injured bac-tera by a membrane filter technique. Appl. Microbiol.23:857-862.

9. Graves, D. C., and . A. Schipper. 1966. Membranefiltration technique for isolating organims from rawmilk of normal udders. Appl. Microbiol. 14:535-38.

10. Halden, H. E. 1957. Methods for count ofmicroorganismsin liquid sugar. J. Am Soc. Sugar Beet Technol. 9:393-399.

11. Halden, H. E., D. D. Leethem, and F. G. Eis. 1958.Developments in methods for microbiological control inliquid sugar. J. Am. Soc. Sugar Beet Technol. 10:138-141.

12. Halden, H. E., D. D. Leethem, and F. G. Eis. 1960.

Determination of microorpnisms in sugar products bythe Millipore method. J. Am. Soc. Sugar Beet. Technol.11:137-142.

13. Hermn, P. H., and H. I Bred.e. 1936. Principles ofthe mathematical treatment of constant pressure filtra-tion. J. Soc. Chem. Ind. London Trans. Commun. 55:1-4.

14. Kirkham, W. K., and P. A. Hartman 1962. Membranefilter method for the detection and enumeration ofSalnoneUa in egg albumen. Poult. Sci. 41:1082-1088.

15. Moros, R. 1957. Determination of yeast in sugar liquidsusing membrane filters. Int. Sugar J. 59:70-71.

16. NuttIng, L A., P. C. Lomot, and F. W. Barber. 1959.Estimation of coliform bactena in ice cream by use ofthe membrane filter. Appl. Microbiol. 7:196-199.

17. Reusse, U. 1959. Quantitative determination of BruceUain cow's milk by means of membrane filters. Bull.W.H.O. 21:782-784.

18. Rodriguez, R. B., and W. G. Walter. 1957. Use of themembrane filter for the microbiological testing of dairyproducts. Sanitarian (Los Angeles) 19:196-197 & 218.

19. Roth, K. 1970. Methodik einer mikrobiologiwchen Kon-trolle bei der Faswasche. Brauwelt 110:567-673.

20. Scheuermann, E. A. 1970. Betriebskontrollen und Pro-zessfltrationen Membranfilter in der Na gamittel-industrie. Er gwirtachaft 10:8404844.

21. 8chiemann, D. A. 1977. Evaluation of the Stomacher forpreparation of food homogenates. J. Food Protect. 40:445-448.

22. Sharpe, A. N. 1978. Some theoretical aspects of micro-biological analysis pertinent to mechanization, p. 19-40.In A. N. Sharpe and D. S. Clark (ed.), Mechanizingmicrobiology. Charles C Thomas, Publisher, Spring-field, Ill.

23. Sharpe, A. N., L P. Diotte, L Dudas, and G. LMfichaud. 1978. Automated food microbiology: poten-tial for the hydrophobic grid-membrane filter. Appl.Environ. Microbiol. 36:76-80.

24. Sharpe, A. N., and A. K.Jao 1972. Stomaching anew concept in bacteriological sample preparation.Appl. Microbiol. 24:175-178.

25. Sharpe, A. N., and G. L Michaud. 1974. Hydrophobicgrid-membrane filters: new approach to microbiologicalenumeration. AppL Microbiol. 28:223-225.

26. Sharpe, A. N., and G. L Michaud. 1975. Enumerationof high numbers of bacteria using hydrophobic grid-membrane filters Appl. MicrobioL 30:519-524.

27. Sharpe, A. N., and G. L Michaud. 1978. Enumerationof bacteria using hydrophobic grid-membrane filters, p.140-153. Chapter 10 In A. N. Sharpe and D. S. Clark(ed.), Mechanizing Microbiology. Charles C Thomas,Publisher, Springfield, Ill.

28. Silliker, J. H.,H P. Andrews, and J. F. Murphy. 1957.A new non-destructive method for the bacteriologicalsampling of meats. Food Technol. (Chicago) 11:317-318.

29. M. L Speck (ed.). 1976. Compendium of methods for themicrobiological examination of foods. American PublicHealth Asociation, Washington, D.C.

30. Tobin, R. S., and B. J. Dutka. 1977. Comparison of thesurface strcture, metal binding, and fecal coliformrecoveries of nine membrane filters. Appl. Environ.Microbiol. 34:69-79.

31. Tuttlebee, J. W. 1975. The Stomacher-its use for ho-mogenization in food microbiology. J. Food Technol.10:113-122.

VOL. 37, 1979


.asm.org/

Dow

nloaded from

http://aem.asm.org/

membrane filtration offoodsuspensions · of membrane filter, flow direction, pressure differential,...

Documents