CA1220075A

CA1220075A - Cheese-flavored substance

Info

Publication number: CA1220075A
Application number: CA000466478A
Authority: CA
Inventors: Donald P. Boudreaux
Original assignee: Borden Inc
Current assignee: Hexion Inc
Priority date: 1983-10-31
Filing date: 1984-10-29
Publication date: 1987-04-07
Also published as: US4675193A

Abstract

CHEESE-FLAVORED SUBSTANCE

Abstract of the Invention A two-stage process for producing a cheese-flavored substance is claimed. A flavor development medium is fermented with a source of lipase/
protease; the source of lipase/protease is inactivated and the flavor development medium is fermented by at least one lactic acid-producing microoganism which is thereafter inactivated. The source of lipase/pro-tease is preferably a microorganism, especially Candida lypolitica ATCC
20234. The cheese-flavored substance produced by the process and foods containing the cheese-flavored substance produced by the process are also claimed.

Description

7~i CEEESE-FLAV~RED SUBST~CE

Field o the Invention The invention relates to a tworst~ge pro oe ss for making a sub-stance having a cheese flavor util-zing a source of lipase, protease or both, at least one strain of lactic acid-prcducing bacteria and a flavor development medium having frcm about 0.3% to about 12~ protein and from 5 about less than 2% to about 85% fat. ~ne flavor development medium con~
taining fats and a sour oe of non-fat nllk solids is fermented for a period of time by the source o lipaseJ~rotease then inactivated The inacti~
vated flavor develoP~ent medium is subsequently fermentr~d by at least one lactic acidproducing microorganim. A relatively lcw solids cheese-1~ flavored substance is produced. The low solids cheese flavor may be con-centra~ed to 95% solids in dry po~der~d form or concentrated into a paste form in a range of ketween 40 and 75~ solids.

Bac~qround of the Invention ~____ The production of cheese by traditionai methods is a time-comsumin~ process. m e startin~ material for such traditional cheeses invariably is whole ~ilX ~r whole ~ilk that has either been augmented with milk fat or treated to separate high milk fat-containing 'ractiGns ~hich are used for the production of cheese frcm relatively lower miIk fat-containing fractions. The producti~n of cheese having gocd flavor by such traditional methods is a ~omplex process that is not well understoodO
~hat is l~derstcod, however, is that the flavors that give the various cheese their distinctive charactertistics are gradually produced during ripening times which may vary from 2-3 rnonths to well over one year at controlled temperatures. ~ecause o the long and exactin~ storage condi-tions or the ripening process such tr~ditionally produced cheeses are an expensive delicacy for a ~small seg~ent of the cheese-consuming population~

An alternative approach to the production of cheeses having a distinctive and pleasing flavor is to c~tain a cheese flavored substance, via vari~us p~o oe ssest ha~ir.g a distinctive cheese flcavor and to add ~liS
Y' j~ "t cheese flavored substance to a relatively young cheese, i~e., one that has not been subjected to a long aying process, or to a cheese that has not developed a distinctive flavorO Such cheese-flavored suhstances, of course, have a more general application in the food industry. They may be used to impart a cheese 1avor to a variety of roods, including: pro-cessed c~.eeses, which include natural cheese, color, salt and emulsifiers;
pro oe ssed cheese fccds which include, in addition to the ingredients of processed cheese, certain optional ingredients such as skim milk, whey, miIk cream, albumin and skim miIk cheese; or processed c~eese spreads which include, in addition to the ingredients in Frocessed cheese food, gums and the like for water retention~ Cheese-flavored substances fur-thermore may be used to impart a distinctive cheese flavor to imitation cheese. Moreover, such distinctive cheese flavors can be used in a num~er of non-cheese type foods. For examDle, they can be used in baked goods such as cheese-flavored crackers, chips, breads, ca]ces, and the like.

In general, cheese-flavored substances may ~e produced by one of t~c methods. In the first method, a traditionally produced unripened cheese having no distinctive fla~7or is subjected to enzymatic digestion.
The digest is combined with various ingredients and added to the focd product to be produced. Although lt is Fossible to produce relatively large amounts of cheese-flavored substances by this general process, it has the distinct disadvanta~e of a relatively expensive starting material.

A second approach tG prcducing cheese flavor is to employ fer-mentative techniques. Cheese-flavored substances produced by processes of ~.his type have almost always used substrates having relatively high mil~
Eat concentrati~ns -- on the order o between 30% and 80% on a solids basis. Usually, such high milk fat substances take the form of cream or butter. In generalt these pro oesses all utilize s~bstrates having rela-tively low protein con oe ntrations, usually not exceeding 3% on a weight/
v~lwme basis. Although it is possible~ using t.~ese convention~l rer~enta-tion processes, to prcduce substances having distinct cheese tastes in a relatively short period of time -- on the order 3f 2 5 days -- these con-ventional techniques have the distinct disadvantage of requiring relative-ly ex~nsive starting materials as substrates. Moreover, these starti~g materials a-e subject to spoilage in short -~eriods or til~e if nct stor~d under proper conditions, usually requirin~ refrigeration. Thus, the sub-strates used in conventional techniques entail considerable e~pense in ac~uisition and storage.

Summary of the Invention The process for producing the cheese-flavored substance a~cord-ing to the instant invention may be briefly described as a two-stage fer-menta~ion of a flavor development medium. m e components of the tw~stage fermentation are a source of lipase or protease or lipase and protease ~herein referred to as lipase/protease), and at least one lactic acid-producing microorganism and a flavor development medium2 The flavor development medium generally will ccmprise fats in a range of from le~s than 0.5% (wt/vol) to about50~ (wt/vDl) and protein frcm about 0.3% (wt~vol) to about 12~ ~wt~vo~ airy substances such as skim milk, whole milk, non-fat dry miIk, whey, whey protein concentrate and the like can ke used. Alternatively or additionally, animal milk (e.g., goat mlLk3, milk fat, or vegetable oils containing fatty acids may also be used.
It is preferred that the source of lipase/~rotease is a lipase/
protease-producing mucroorsanism. m e lipaseAprotease-producing mirc-organism(s) may be produced in ~o steps~ In the first step of the tw~
steps, the inoculum develoFment step, the lipase~rotease-producinq organ-ism(s) is grown for a period of time in aerobic ~onditions in a ccmpleteinoculum develo~nent medi~m. In the second of the two steps an alio~uot of the inoculum development medium containing the lipase/protease-producin9 organism(s) is placed in a first starter development medium and is cul-tured under aerobic ccnditions for a period of time.

A~ter the first starter development medium containing the lipase~protease-producing microorganism has reached a predetermlned growth point, the first stage of the two-stage fermentation is initiated by inoculating an aliquot of the lipase/protease-producing microorganism in the first starter development medium into the fla~or development medium.
~he flavor development medium is fermented for a period of time and the lipase~protease producing microorganism and enzymes are inactivated to terminate the first stage of the twc-s~aqe ~ermenta~ionO At the sam2 ~, time, at least one lactic acid~producing tnicroorganism is grcwn anaerobic-ally in a second starter development medium for a period of time. The second stage of the two-stage fermentation is initiated by inoculating an ali~ot of the second starter development m~dium containing the lactic acid-producing microorganisms into the previously inactivated flavor development medium. After a period of time, the medium is again heat-inactivated. The process is descri~ed in further detail hereinbelcw.

Detailed Description of the Invention m e process according to the subject invention is a two-s-tage fermentation of a flavor development medium utilizing a source of lipase~protease and at least one lactic acid-producing microorganism. The sources of lipa~e and protease may be any purified or partially purified preparations of lipase and protease. Such purified and partially purified sources of enzyme are readily available co~nercially. Alternatively, the process according to the inver.tion is a two-stage fermentation of a flavor development medium by, as the source of lipase/~rotease, a lipase/pro-tease producing microorganisn and at least one lactic acid-prcducin~
microorganism. The term "lipase/~rotease-producins microorganism" as used herein is intended to include: any lipase-producing microorganism, pre-ferably any extra oellular lipase-producing microorganism; any pL-otease-producing microorganism, preferably any extracellular protease-producing mQcroorganism; a ccmbination of lipase-producing microorganism and protease-producing microorganism; or any lipase- and proteaseproducing mlcroorganism. The choice of lipase~protease-producing microorganism will depend upon the contents of the flavor development medium.

The process according to the invention al o encompasses a two-stage fermentation of a flavor develoFment ~edium in which the source of lipase/~rotease is a purified or partially puriied preparation of lipase~
protease and a lipase~protease-producing microorganism. Thus, within the scope of the invention are fermentations of a flavor development medium in which the source of lipase/protease may be a purif ed or partially puri-fied protease and a lipase-producing microorganism, a purified or partial-ly purified lipase and a protease-~rcducing microrcganism; or a lipase-and protease-producing microorganism supplemented with a purified or partially purified lipase or protease or koth.

~2~

Preferably the lipase~protease-producing microorganism will be nonpathogenic, and particularly nonpathogenic to man. In particular, microorganisms such as -~ Penicill~m (especially Pennicillum roqueforti), , _ _ Oidvm lactis, ~ (especially ~ butaryi), Micrococcus and Candida (especially Candida liPolytica) are suitable microorganisms.
Preferred in the process according to the present inventlon is Candida and in particular, Candida lipol tica deposited in the American Type Culture Collection, 12031 Parklawn Drive, Rockville, L~aryland 20352-1776 USA under the accession num~ers ATCC 8661, ATCC 20320 and ATCC 20324.
Especially preferred is Candida ~ ATCC 20324.

As mentioned above, at least one lactic acid producing micro-organism is used in the twc~stage fermentation according to the invention.
Lactic acid producing microorganisms suitable for this purpose are desir-ably dairy strains of lactic acid produ~ing organisms. Dairy strains of lactic acid producing microorganisms include the genuses ~
(S.), Lactobacillus (L.~, Leuconostoc (Le.), Micrococcus (M.), Citrobacter (C ) and Brevibacterium (B ). Within the genus ~ , S.
cremoris, S. lactis, S. thermo~hilous and S~ diacetilactis are examples.
Within the genus Lactobacillus, L. casei, L. bulgaricus, L. acidoPhilousr L. delbrueckil, and L. helveticus are examples~ Within the genus Leuconostoc, Le. cremoris, Le. dextranicum and Le~ citrovorum are examr _ ~
ples. Within the genus Mlcrococcus, M ca~elvtics9 M oonqlomeratus and M. freudenreichii are exa~ples. Within the genus Citrobacter, C.
.~
intermedii and C. freudii are examples. Within the genus Brevibacterium, B linens is an example.
_ _ .

It is preferred in the process according to the invention~ to use two lactic acid-producing microorganisms, and although any t~o of the above listed lactic acid-producing microorganisms are usable, S lac~is and L. casei are preferred. Particularly preferred for the S lactis is S. lactis strain C2 kindly furnished by W.E. Sandine of Oregon State University at Corvallis, Oregon. Also preferred is L casei, deposited in the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland, under ac oe ssion number ATCC 39392.

~2~ 7~

The lipase,~rotease-prcducing microorganism(s) and lactic acido producing microorganisms used in the two-sta~e fermentation according to the invention are separately developed for subsequent ir.oculation into the 1avor development medium. The lipase~rotease-producing microorganism(s) i developed for inoculation in two stepsO Preferably this development is carried out under aerobic conditions. In the first of the t~D steps, the lipase/protease-producing microorganism(s) is inoculated into an inoculum development medium which is a complete medium suitable for grcwth of the lipase/protease-producing microorganism(s). Preferably, the ocmplete medium will contain all the substances necessary for vigorous and sus-tained growth of the lipase~protease-producing microorganism(s)O In general, such a mediwm will be a standard bacterial culture nedium supple-mented with yeast extract (Y~. Examples of such media included but are not limited to Nutrient Broth (produced by Difco), Yeast Morphology Broth (Difco), Peptone (Difco), and Tryptocase Soy Broth (TSB) (Bethesda Biological Laboratories, hereinafter BBL). Yeast extract is available frcm Difco and Stauffer Chemical Company (hereinafter SCC) under the tradename KAT. Preferably Car.dida ~ is inoculated into a three __ percent (~) TSB 1~ Y medi~m and is grcwn aerobically for a period of time and at a temperature sufficient to yield a predetermined cell titre, gen-erally between la6-109 cells/~illiliter (ml). A suitable temperature will range between 23-35C. 30C is preferred, at which temperature the Eirst step of development will require from about 16 to about 18 hoursr In the second step of develoFment of the lipase/~rotease-producing microorganism(s) for inoculation, an aliquot of the liF~ase/
protease-producing microorganism(s), obtained at the end of the first step is inoculated into a first starter development medium and is cultured aerobically. m e starter developm~ent medi~m may be any substrate that will support the vigorous grcwth of the lipase~rotease-producing micro-organism(s) and the production of lipase or protease or both lipase andprotease. Such substrates include whole milk, non-fat dry milk (NFDM) resuspended in water, fluid skim milk, whey, concentrated whey, or dried whey (such as Teklac~) (Fonnost) resuspended in ~ater or dairy starter culture medium such as Insure~ (Hansens, Milwakee, wisconsin)O The fore-going starter development substrates may ~e used sin~ly or in any oombina-tion. I~ NF~M is used, it is preferably resuspended in water to about 10%

~7~

~wt/vol %). If concentrated whey or dried whey is used as the substrate, it is diluted or resuspended at a concentration of about 7% to ab~ut 15%
(wtho~

m e inoculated first starter development medium is incubated at ab~ut 30C for 12 hours or until cell titres reach frcm a~out 1o8 to about 5 x 1o8 cells/~l. During the incubation period the pH rises from about 6.4 to about 7O0. During incubation of the first starter develop-ment medium, lipase, protease or lipase and protease activity is moni-tored. Lipase activity in a range of about 11 units ~U)/~l to about 30 U/~l is desirable. Preferably the lipase activity will be between about 20 U/~l and 26 U/nl as determined ky usin~ ~ipase Test Kit No. 800B, available from Si~ma Biochemicals, St. Louis, Missouri.

Protease activity during the first starter development step is monitored by spectrophotot~etrically measuring the absorban oe of soluble azure blue dye liberated by proteolysis of hide azure powder ~Calbiochem Catalogue #37716) by an aliquot of the first starter development medium.

Aliq~ots of the first starter develoFment m~dium that has incu-bated to the extent desired are transferred into a flavor development medium which is described further hereinbelow. In general, the transfer of the first starter develoFment medium containinq the lipase/prote~se-producing microorganism(s~ will be in a range of from about 3% to a~out 35~ (wt~wt) of the flavor development medium. An inoculum of the first starter development m~dium of about 10% (wt/vol) is preferred.

m e lactic acid-producing microorganisms are separately cultured ~5 in a second starter developnent medium which may be any dairy-starter medium suitable for the vigorous growth of the lactic acid-producing microorganisms. Examples of such dairy starter m~dia incluie a product sold under the name In-Sure~ (Hansen's, Milwa~ikee~ ~isoons~n 53214), Funnel Grade (Miles Labs., M~rschall Div., Wisconsin~, MSM (Miles Labs.
Marschall Div., Wlsconsin), One-2-one (Miles Labs., Marschall Div., Wisconsin), T~S.S. ~Dederich Corp., Germantown, Wisconsin), Phage Stat * *
Complete (Pizerl New York, New York) and Phase 4 (Gallcway Wes., Fond Du Lac, Wisconsin).
* Trademark :~.

~2~

The lactic acid producing microorganisms are inoculated into the second starter development medium frcm slants and the medium is incubated for a sufficient period of time, generally between about 12 and 30 hours, at a sufficient temperature, generally in a range of between about 23 and about 35C to bring the second starter development medium to a p~ from about 5.3 to about 5.5. Preferably, the incubation proceeds for about 20 hours at 30C at which time the cell titre for each of the lactic acid-producing microorganisms used will generally equal or exceed 1o8 cellsJ~l.
m e duration of and temperature for the incubation of the second starter development medium can be varied as necessary so that the first starter develoFment medium and second starter development medium are ready for transfer into the flavor development medium when needed~

In general, the transfer of the second starter development medium containing the lactic acid producing microorganisms ~ill be in a range of from about 0~5% to about 5% (wt/vol). ~n inoculum of the second starter development medium of about 1 to 2% (wt~vol) is preferred.

Generally, the flavor develoFment m~dium is sequentially inocu-lated with transfers of the incubated first and second starter development media with an inactivation step between the tw~ transfersO m us, in the first stage, the flavor developmen~ medium is inocula~ed with a transfer of the first starter development medium containing the lipase/protease-producing microoganism. After a period of time the first stage is termin-ated by heat inactivation of the lipase~rotease prior to àddition of the lactic acid-producing microorganisms. In the second stage, the heat inactivated flavor development medillm is inoculat~d with a transfer of the second starter development medium containing the lactic acid-producing microorganisms. After a period of time the second stage is also termin-ated by heat inac~ivation.

The flavor development medium may be c3mprised of sklm milk, defined as milk having less than 2% milk fat, whole milk, non-fat milk solids including non-fat dry milk, whey and whey protein concentrate~
butter, cream or any other dairy product acceptable by the Food and Drug Administration Standards for the making of pro oe ssed cheese food. Fur-thermore, if the cheese-flavored substance is to be used in imitation .

cheese,vegetable oils (preferably those incl~ding fatty acids of 10 car-bons inclusive or less, for example c wonut oil, palm oil ar.d the like), casein and animal milk fats may be used.

The flavor develoFment medium may have a fat content of frcm less than 2% to 50% on a weight/volume percent basis. m e protein content will generally range from about 0.7% to about 3~ (wt~vol) protein.
Preferred is a flavor development medium ccmprising fats and non-fat milk solids.

In the first stage, the flavor development nedi~m is fermented at a temperature and for a period of time sufficient to reduoe the pH of the flavor develoFment medium to p~ between about 4.5 and about 5O5. If a temperature of 3CC is used the fermentation time will range between about 8 and about 48 hours depending on the amount of fat in the fat in the flavor develoFment medium. Lower fermentation temperatures require con-comitantly longer periods of time. If the flavor development medium has arelatively high butter fat content (S0% w/v), the butyric acid con oe ntra-tion of the flavor develo~ment medium may be nitored instead of or along with pH. A butyric acid con oe ntration reachin~ from akout 0.7~ to 1.15%
(wt~wt) is acceptable. A butyric acid concentration of about 1% is pre-ferred.

When the proper p~ or butyric acid concentration is reached, thefirst stage of the 1avor development medium fermentation is terminated by heat inactivation at a temperature sufficiently high to inactivate any microganism~s), protease, lipase or all three. Generally, raising the temperature to about 85C for about 45 minutes is sufficient. Alterna-tively, the above-mentioned microorganisms and enzymes may be inactivated by exposing the flavor development nedium to very high temperatures exceeding 100C for short periods of time.

The second stage of the flavor development medium fermentation may be terminated, after the desired pericd of time in a sLmilar manner.
In general, the second stage of the flavor developnent medium fermentation will reguire between 3.5 and ~0 nours ~t temperatures between 4C ar~
about 35C.

A cheese-flaJored substance is thus formed that may be spray dried, freeze-dried, concentratedr or kept in unconcentrated form. If spray dried, a solids content of about 95% is desirable. If concentrated, a paste consistency of from about 40% to about 60~ solids is desirable.
Such paste form of the cheese ~lavored substan oe may have a fat content range of frcm less than 2~ to a~out 85%.

The spray dried product will have a fat content range of from less than 2% to about 55~ of the sollds and a protein content range of from about 1% to about 40~ of the solids. In general, a protein content of from about 21~ to a~out 38% and fat cGncentration of frcn less than about 2% to 40% is desirable in the spray dried prcduct~

m e cheese-flavored substance produced by the above-descri~ed process may be used as a cheese flavor in processed cheese foods, and pro-cessed cheese spreads or may be added to various foods including imitation cheese and cheese~flavored snack foods~ In addition, butter fat may be added to the cheese-flavored substan oe before blending in the various foods. The pro oess for producing the cheese-flavored substance and the cheese-flavored substance produced thereby will be better understood from the following examples which are intended by the inventor to ~e exemplary only and non-lLmiting.

EXAMPLE I
_ lipolytica ATCC 20324 was inoculated frGm a slant into an inoculum development medium ccmprised of 3~ qSBy 1% Y and was incubated for 40 hours at 30~C.

Four liters ~l) of a first starter development medium ccmprised ~5 of 20~ NFDM (Difco) was divided into t~o 2 liter portions and each was placed in a 6 1 flask and autoclaved for 20 minutes. After cooling, each of the flasks containing the first starter development medium was inoculated with a 10% transfer of incubated inoculum develoFment medium containing C. liF~lytica 20234 and was allowed to incubate for 40 hours at 30~C with rotary agitation at 200 rFm. A flavor develoFment medium ccn-prising 750 g of salted butter was heated to 85C for 30 minutes in a 2 1 flask, cooled, inoculated with 450 grams (g) of the incubated first starter development medium, and incubated for 48 hours at 30~C with rotary agitation at 200 rFm. At the end of the 48 hour Feriod the fIavor devel-opment ~edium was removed from the flas~ and heat inactivated at 85C for 30 minutes~

A second starter develoFment medium! co~prising lactose broth supplied ~y Dirco, 3.0 g/l beef extract, 5.0 g/l bacto-peptone, 5.0 g/l bacto latose and having in addition 5.0 g/l lactose and 2~5 g/l casein was inoculated with S. lactis C~ and incubated with aeration at 30C ~or 48 _ __ hours with rotary agitation at 200 rpm~

m e previously inactivated flavor development medium was cooled to 30C and inoculated with a 2~ transfer of S. lactis C2 frcm the ircu-bated second starter develoFment culture, sealed with parafilm and incu-bated at 30C for 3.5 hours with rotarv agitation at 200 rpm. At the end of 3.5 hours the flavor development medium was divided into 6 equal por-tions and the bottles were cold stored at 4C for 72 hours. At the end of 72 hours, half the samples were heat inactivated at 95C for 30 minutes.
The samples were then ripened at rocm temperature for 3 days.

rme products so produced were designated lH (heated) and 1NH
(non-heated).

EXAMPLE II
A second fermentation product was prepared in the same manner as in Exan~le I except for the follcwing. A second starter development medium comprising 10% skim milk (Difco) and 0.5~ yeast extract was inocu-lated with S. cremoris 134 and was incubated under the same conditions as Example I hereinabove.

m e previously inactivated flavor development medium was cooled to 30C, and inoculated with a 2~ transfer of the second starter develop-ment medium. m e sample was thereafter treated as in Example I herein-above. The t~ samples so produced were designated 2~ and 2NH.

E~IPI E III
A third fermentation product was prepared as in Example I except for the following.

Five hundred milliliters (ml) of a second starter development medium having the sa~,e compositicn as the second starter development medium of Examæle I was inoculated with Lactobacillus casei TA101, AI~C
39392, and was grown at 30C with rotary shaXing at 170 rFm for about 18 hours in a sealed screwtop flask.

The previously inactivated flavor development medium ~was inocu-lated with a 1~ transfer of Lc casel TA101 A~CC 39393 and a 1% transfer of S. lactis C2 produced as in Example I. The sample was thereafter treated as in ~xample I hereinabove. The two samples so Froduced were designated 3H and 3NH.

EXP~IPLE IV
A fourth fermentation product was prepared as in Example I
e~cept for the follawing.

One hundred ml of a second starter development medium comprising 10~ NF~M or lactose broth (described in Example I here~nabove) was inocu-lated with S lactis diacetilactis ATCC 11007 and incubated in a second flask at 30C under aerobic ~onditions with rotary agitation at 220 rpm~

m e previously inactivated flavor development medium was inocu-lated with a 1% transfer of S. lactis diacetilactis ~CC 11007 and a 1~~ransfer of SO lactis C2 produced as in Example I hereinabove. ~he two samples so produced were desi~nated 4H and 4NH.

~:E V
A fifth fermentation product was prepared as in Example I except for the following.
The previously inactivated flavor develoFment medium was inocu-lated ~with a 1% transfer of S. lactis produced as in Ex~ple I hereinabove and a 1% transfer of S cremoris 134 produced as in~Example II herein above. m e sample was thereafter treated as in Example I hereinakove.
The t~o samples so produced were designated 5H and 5NH.

~2~

~X4~PLE VI
A sixth fermentation product was prepared as in Example I except for the following.

The previously inactivated flavor develoFment medium was inocu-lated with a 1% ~ransfer of S. cremorls 134 produced as in Example II
hereinabove and a 1% transfer of L~ casei TA101 ATCC 39392 produced as m Example III hereinabove. m e t~o samples so produced were designated 6H
and 6NH.

Exaxpr,r bII
A seventh fermentation product was prepared as in Exa~ple I
except for the following.

m e previously inac~ivated flavor development medium was inoculated with a 1% transfer of S. cremoris 134 produced as in Example II
hereinabove and a 1% transfer of S. lactis diacrdtilactis produced as in Example IV hereinabove. m e sample was thereafter treated as in Example I
hereinabove. m e two samples so produced were designated 7H and 7NH.

EXAMPLE VIII
The samples produced in Exa~ples I-VII hereina~ove were evalu-ated for taste in a processed cheese spread loaf. The processed cheese spread loaf comyrised 64~ barrel American cheese, which is an unaged cheese. No aged cheese was used in the processed cheese spread loaf. The ermentaton products were blended into the prccesses cheese spread loaf at ~0 2.5% (weight~wei~ht) of the loaf. The evaluation of the samples repcrted below records taste impressions Lmparted to the cheese spread loaf by the fernentation products additional to any flavor inherent in the cheese spread loaf.

Sa~ e No. Ccmments 1NH ~eak, clean (slight liFolytic), slight-medium lipolytic odor. Slight cheese slight lipolytic flavor.
~NH Good overall, very slight bitter, medium intensity.
Medium lipolytic odor. Stron~ lipoly.ic flavor note.

7~

3NH ~erate intensit.y, g~d lipase level. Slight lipolytic odor. Sli~ht to medium cheesy. ~eak cheese aftertaste~
4NH Weak, clean. Slight-mediu~ odor. F~irly ~ell rounded~
51ight li~olytic flavor. Slight-medium cheese flavor.
SNH L~edi~ intensity1 clean, gocd lipase, sli~ht sour.
~edium lipolytic odorO Slight-~edl~m cheesv flavor.
Slight lipoly.ic ba~cground.
6NH Gccd, high cheese intensity, good lipase level. Strong lipolytic Gdor. Medium plus flavor level.
7~1 Bitter, siight soapy to unclearn. Highest flavor inten-sity. Strong lipolytic odor. Similar to 6 N~.
lH Sli~ht soapy. Slight lipolytic odor. Very sligh~ lipo-lysis, mild to medium cheesy 1avor.
ZH Slight soapy. Sligh~-medium lipolytic ~dor. Medium-medium plus flavor level. Lipolytic flavor pronounced~
3H S oth, creamy, lower lipase. Slight lipolytic odor.
Medium cheesy flavor~
4H Similar to 3H, slight higher lipase. VerY slight lipo-lytic odor. Similar to 3H.
5H Sour~ slight soapy. Slight lipolytic odor. ~dium flavor level.
6H Sli~ht bitter, medilsn li~ase flavor. ~ery slight lipo-lytic odor. Slight-medium cheesy flavor. Lipolytic.
7H Very sli~ht bitter, otherwise smcoth, balanced~ Slight to medium lipolytic odor. Slig~lt-medlum cheese flavor.

Claims

WHAT IS CLAIMED IS:

1. The method of producing a cheese flavored substance comprising the steps of:
(a) providing a dairy flavor development medium;
(b) fermenting said flavor development medium with at least one microorganism source of lipase/protease producing at least one enzyme selected from the group consisting of lipase and protease;
(c) inactivating said source of lipase/protease;
(d) incubating said fermented flavor development medium with at least one lactic acid-producing microorganism; and (e) inactivating said lactic acid-producing microorganism.

2. The method of Claim 1 wherein said microorganism is selected from the genus Candida

3. The method of Claim 2 wherein said Candida is Candida lipolytica ATCC 20324.

4. The method of Claim 1 wherein said at least one lactic acid-producing microorganism is a dairy strain of microorganism.

5. The method of Claim 4 wherein said dairy strain of micro-organism is selected from the group of genuses consisting of Streptococcus, Lactobacillus, Leuconostoc, Micrococcus, Cirobacter and Brevibacterium.

6 . The method of Claim 5 wherein said Streptococcus is selected from the group consisting of S. cremoris, S. lactis, S. thermophilous, and S. diacetilactis.

7 . The method of Claim 5 wherein said Lactobacillus is selected from the group consisting of L. casei, L. bulgaricus, L. acidophilous, L.
delbrueckii, L. helveticus.

8 . The method of Claim 5 wherein said Leuconostoc is selected from the group consisting of Le. cremoris, Le. dextranicum and Le.
citrovorum.

9 . The method of Claim 5 wherein said Micrococcus is selected from the group consisting of M. caselytics, M. conglomeratus and M.
freudenreichii.

10 . The method of Claim 5 wherein said Citrobacter is selected from the group consisting of C. intermedii, C. freudenii.

11. The method of Claim 1 wherein said at least one lactic acid-producing strain of microorganism comprises two lactic acid-producing microorganisms.

12. The method of Claim 11 wherein said two lactic acid-producing microorganisms are selected from the genuses consisting of Streptococcus, Lactobacillus, Leuconostoc, Micrococcus and Brevibacterium.

13. The method of Claim 12 wherein said Streptococcus is selected from the group consisting of S. cremoris, S. lactis, S.
thermophilous, and S. diacetilactis.

14 . The method of Claim 12 wherein said Lactobacillus is selected from the group consisting of L. casei, L. bulgaricus, L.
acidophilous, L. delbrueckii, L. helveticus.

15 . The method of Claim 12 wherein said Leuconostoc is selected from the group consisting of L. cremoris, L. dextranicum and L. citrovorum.

16. The method of Claim 12 wherein said Micrococcus is selected from the group consisting of M. caselytics, M. conglomeratus and M.
freudenreichii.

17. The method of Claim 12 wherein said Citrobacter is selected from the group consisting of C. intermedii, C. freudenii.

18. The method of Claim 12 wherein said two lactic acid-producing organisms are Lactobacillus casei and Streptococcus lactis C2.

13. The method of Claim 1 wherein said flavor development medium comprises at least one substance selected from the group consisting of skim milk, whole milk, non-fat dry milk, whey, whey protein concen-trate, dried whey, butter, cream and milk fat.

20. The method of Claim 19 wherein said flavor development medium comprises at least one substance selected from the group further consisting of vegetable oils including fatty acids of 10 carbon atoms inclusive or less, animal milk, animal milk fat and casein.

21. The method of Claim 1 wherein said incubating step is car-ried out until the pH of said flavor development medium reaches from about 4.5 to about 5.5.

22. The method of Claim 21 wherein said pH is about 5.

23. The method of Claim 1 wherein the duration of said first fermenting stage is about 6 to about 48 hours.

24. The method of Claim 1 wherein the duration of said first fermenting stage is about 48 hours.

25. The method of Claim 1 wherein the duration of said second fermeting stage is about 3.5 to about 80 hours.

26. The method of Claim 1 further including after the second ferementation stage the step of concentrating the fermented flavor devel-opment medium.

27. The method of Claim 26 wherein said fermented flavor devel-opment medium is concentrated to a solids range of from 30-95% solids wt./wt. %.

28. The method of Claim 26 wherein said fermented flavor devel-opment medium is concentrated to a paste with a solids range from about 40 to about 60% wt./wt.

29. The method of Claim 26 wherein said fermented flavor devel-opment medium is concentrated to a solids content of about 95% solids wt./wt.

30. The method of Claim 26 wherein said concentrating step is spray drying.

31. The method of Cliam 26 wherein said concentrating step is performed after the second inactivating step.

32. The method of Claim 26 wherein said concentrating step is performed before said inactivating step.

33. The method of Claim 26 wherein said concentrating step is performed simultaneously with said second inactivating step.

34, The method of Claim 1 wherein said flavor development medium comprises fats in a range of from less than about 0.5% (wt/vol) to about 50% (wt/vol) and protein in a range of from about 0.3% (wt/vol) to about 12% (wt/vol).

35. The method of Claim 1 wherein said flavor development medium comprises less than about 2% fat and about 5% protein.

36. The method of Claim 1 wherein said flavor development medium comprises about 0.3% protein and about 50% fat.

37. The method of Claim 1 wherein said flavor development medium comprises about 3% protein and about 6.5% fat.

38. A cheese flavored substance produced by the method of Claim 1.

39. A cheese flavored substance produced by the method of Claim 1 further including butter fat added thereto.

40. A cheese flavored substance produced by the method of Claim 2.

41. A cheese flavored substance produced by the method of Claim 2 further including butter fat added thereto.

42. A cheese flavored food product comprising the cheese flavored substance of Claim 38 and processed cheese, processed cheese food processed cheese spread or other non-cheese type foods.

43. A cheese flavored food product comprising the cheese flavored substance of Claim 40 and processed cheese, processed cheese food, processed cheese spread or other non-cheese type food.