Indian Journal of BiotechnologyVol 2, April 2003, pp 220-226

Apple Pomace Utilization for the Production of Baker's Yeast: Effect of SubstrateConcentrations and Growth Stimulators

V K Joshi* and Shashi BhushanDepartment of Postharvest Technology, Dr Y S Parmar University of Horticulture & Forestry. Nauni, Solan 173230, India

Received 7 June 2002; accepted 17 October 2002

Apple pomace powder showed higher percentage of fermentable sugar (to that of total), titratable acidity andhigher crude protein than molasses and jaggery. The composition of the extracted/diluted medium was in consistencewith the original substrates. Among the different total sugar concentrations tried (5, 10 & 15%),5% was optimumfor higher yeast biomass production. Comparing glucose with apple pomace extract (APE), molasses and jaggery,glucose gave higher fermentation efficiency followed by APE, which was found superior with respect to highercellular yield coefficient and lower ethanol production. Addition of growth stimulators (vitamins & minerals)increased efficiency of baker's yeast in both ways i.e. to respire or to ferment the available sugar. The differentcarbon sources behaved similarly with respect to the addition of growth stimulators (minerals, vitamins and theircombinations) except APE medium, which exhibited a non-significant effect. Supplementing the APE medium withsuch growth stimulators thus, is not required to produce baker's yeast.

Keywords: apple pomace, molasses,jaggery, Baker's yeast

IntroductionOut of the total apple production, around 30% is

processed into different products such as juice andconcentrate, but wine, cider, canned slices, vinegar,cubes, etc. are also made occasionally (Sharma &Joshi, 2002). Apple pomace, a left-over residue afterjuice extraction containing peel and remaining solidparts, is estimated to be about 20-30% of the totalprocessed fruits (Sargent et al, 1986; Wang &Thomas, 1989; Joshi, 1998a; Maini & Sethi, 2000). Itis a rich source of carbohydrates, dietary fibres,minerals and vit-C. It is biodegradable and usedfermentatively (Smock & Neubert, 1950; Kennedy,1994, Joshi et al, 1996, Devarajan, 1997; Joshi et al,1998). It has shown its potential as a substrate to growmicrobial cells like other wastes (Joshi et al, 1998).Apple pomace, being a low bulk and high valueproduct, holds great promise for baker's yeastproduction. Traditionally, molasses (a waste fromsugar industry) is the only substrate utilized toproduce baker's yeast but from the last decade or soits utilization is becoming unattractive due toimproved methods of high sucrose recovery and lowerfermentable sugar concentration. Moreover, molasses

*Author for correspondence:Tel: 01792-52410; Fax: 01792-52242E-mail: vkjoshipht@rediffmail.com

medium requires heavy supplementation with growthpromoting substances such as mineral and vitamins,besides its clarification and pretreatments beforeutilization, thus adding to the production cost.Production of baker's yeast from apple pomace is aworthwhile proposition. The suitability of applepomace for the production of baker's yeast,optimization of sugar concentration in the fermentingmedium, growth stimulators as well as effect ofvarious carbon sources is presented in this paper.

Materials and MethodsRaw Materials

Apple pomace. The fresh apple pomace, collectedfrom departmental fruit processing unit, wasmechanically dehydrated at 60±I°C till the moisturecontent of dried apple pomace reached 4±1 % in about8-10 hrs. The dried apple pomace was also procuredfrom Himachal Pradesh Horticultural ProduceMarketing and Processing Corporation's ProcessingUnit, Parwanoo (HP), to meet the experimentaldemand during the off-season.

Glucose, molasses and jaggery. Commercial gradeglucose, molasses and jaggery were procured fromlocal market (Solan) to determine the efficacy of thesubstrates.

Growth stimulators. Vitamins (biotin, thiamin,pyridoxin, nicotinic acid and pantothenic acid) and

JOSHI & BHUSHAN: APPLE POMACE FOR BAKER'S YEAST PRODUCTION

minerals (di-ammoniurn orthophosphate, sodiummolybdate, di-hydropotassium phosphate, calciumchloride, sulphates of magnesium, zinc, copper, ironand manganese) were used as growth stimulators formedia supplementation.

Media PreparationApple pomace extract (APE). The dried apple

pomace was ground into fine powder, which wasdiluted with water (1:6) and boiled for half an hourfollowed by filtration to yield APE (65.7%). The APEwas then sterilized at 121°C for 20 min in a 5Lconical flask as per the standard procedure. Themedium was enriched (Joshi et al, 1998) with yeastextract (0.3%), beet extract (0.3%), peptone (0.5%)and ammonium sulphate (1.8%).

Molasses and jaggery medium. The molasses andjaggery were diluted with water (1:9) followed byfiltration. The media were sterilized similar to APE.

Growth stimulator solution. The vitamin andmineral solution, used as growth stimulator, wasprepared by dissolving them separately as stocksolution followed by sterilization. Stock solutions ofvitamins and minerals of specified composition wereprepared for production of baker's yeast (aura, 1974;White, 1954; Frey et al, 1936; Harrison, 1967).Growth stimulators added to the media are: vitamins-D-biotin, 0.125; thiamin-HCI, 5.0; pyridoxin-HCl,6.25; nicotinic acid, 5.0; and D-pantothenate-calcium,6.25 mgl."; and minerals- (N~)2 HP04. 0.60; Na2Mo04 2H20, 0.033; KCI, 0.12; KH2 P04, 0.50; CaCh2H20, 0.10; MnS04 H20, 0.006; ZnS04 7H20, 0.095;CUS04 5H20, 0.125; FeS04 6H20, 0.15; and MgS04H20, 0.50 gL·'.

Inoculum PreparationBaker's yeast, Saccharomyces cerevtsiae var.

diastaticus, used as inoculum, was procured from thelocal market available under the brand name of'Sailors' Active Dry Yeast, Sailor's Corporation, NewDelhi. The culture was maintained on yeast maltextract agar slants and kept under refrigeratedconditions. From the slant culture of baker's yeast,inoculation was made in yeast malt extract brothincubated for 24 hrs at 30°C and then transferred torespective medium and incubated at the sametemperature for another 24 hrs. The inoculation ofrespective medium was done @ 1% to initiate thefermentation.

Fermentation ProcessUp-streaming processes. Screening of media was

done in batch culture system using lL conical flasks

221

at room temperature (25±2°C). The respective mediawere sterilized, separately. The addition of sterilizedgrowth stimulators was done under aseptic conditions,as per the composition. Thereafter, inoculum @ 1%was added to the respective media and kept forfermentation on shakers with continuous shaking atambient temperature (22-25°C).

Down-streaming processes. Harvesting of theproduced yeast biomass was done manually. Theyeast containing the broth was centrifuged at 4,000-5,000 rpm followed by washing with sterilized waterand then again centrifuged at 10,000 rpm for 10 min.The yeast cream was then filtered through a Whatmanfilter paper (No.1) to yield a compressed cake (wetcompressed yeast), which was wrapped in wax paperand stored at 0±1 "C.

Experimental LayoutSeparate experiments were conducted for the

optimization of sugar concentration and observing theeffect of growth stimulators on growth of baker'syeast, under batch culture system. Mediumoptimization with respect to different initial sugarconcentrations i.e. 5, 10 and 15% was carried outusing different substrates, glucose, APE, molasses andjaggery. The procedure with regards to inoculumpreparation and fermentation conditions was followedas per the details given in earlier section. Thedifferent sugar concentrations were raised with theaddition of commercial grade glucose in case of APEwhile in others only dilution was done. Theobservations were recorded, initially and after the endof fermentation (i.e. up to 24 hrs), for sugarconcentration, ethanol production, pH and cellularyield coefficient (CYC). In case of effect of growthstimulators and substrates on the growth of baker'syeast, all the four types of substrates were evaluatedeach at the sugar concentration optimized in earlierexperiment. With each substrate, four types of mediawere prepared as such or were supplemented withvitamin, mineral or both as per the composition earlierdescribed. The fermentation was carried out asdescribed in an earlier section. The details of thetreatment combinations were: L" Control; L2,Minerals; L3, Vitamins; and, L4, Minerals &Vitamins, each for individual medium.

AnalysesDetermination of reducing sugars was based on

Nelsen-Somogyi method, while for totalcarbohydrate, the phenol sulphuric acid method was

222 INDIAN] BIOTECHNOL, APRIL 2003

used (Sadasivam & Manickam, 1996). Titratableacidity was determined with standard method andexpressed as per cent malic acid. Apple pomacepowder was diluted appropriately. The pH was takenwith ELTOP 3030 pH meter, after calibrating it withbuffer solutions of pH 4 and 7 (Ranganna, 1986).

eye. It was calculated on the basis of grambiomass produced during fermentation from the totalsubstrate (as glucose equivalent used) consumed(Aiba et al, 1976; Wang et al, 1977; Stanbury et al,1997), and expressed as:

CYC = biomass produced I sugar consumed

Total biomass yield. Total biomass yield wascalculated on the basis of cumulative gram biomassproduced during fermentation from total medium fedat that particular time as detailed below:

x = X, X Vt, whereX = total biomass yield (g)X, = biomass at time t (g)V, = volume of medium in the fermenter at time t (ml)

Statistical analyses. Data from physico-chemicalcharacteristics of substrates, fermentation process andbaker's yeast were analysed with completelyrandomized block design, wherever applicable.

Results and Discussion

Effect of Sugar ConcentrationThe results (Table 1) revealed that the mean

reducing sugar consumed was significantly lowest at5% initial sugar concentration (lSC) followed by 10%and 15%. With the increase of sugar content from 5 to15%, the percentage of sugar consumption to that oftotal sugar declined. The pattern of total sugarconsumed was similar to that of reducing sugarconsumed by the yeast. A lower pH was recorded athigher sugar concentration (4.47 at 15%). Thedecrease possibly be due to the production of moreorgamc acids at higher sugar concentration duringfermentation (Amerine et al, 1980). The maximummean ethanol content (5.67%) was recorded at 15% ofISC as compared to 10 and 5%. In case of CYC, theISC of 5% recorded significantly higher (0.33 gigsugar consumed) than at 10 or 15%. The higher ISC(10 or 15%) failed to produce higher biomass. It isamply clear from the trend (Fig. 1) that as the ISCincreased from 5 to 15%, the quantity of sugarutilized increased along with ethanol production. But

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opposite trend was observed in CYe. It clearlyindicates that increase in sugar concentrationincreased the amount of ethanol content, but reducedthe CYe. The production of more alcohol byconsuming higher quantity of sugar is attributed to thefact that under anaerobic conditions glucose is moreefficiently converted into ethanol and CO2 than inaerobic mode by the yeast (Amerine et al, 1980).Further, the higher sugar concentration might haveexerted crab tree effect (Franz, 1964), which is knownto decrease the levels of enzymes of electron transport'system and citric acid cycle resulting in a shift fromrespiration to fermentative metabolism of yeast, thus,producing more ethanol.

The results pertaining to the effect of differentsubstrates (Table 1), irrespective of ISC, showed thatmean sugar consumption was significantly higher inglucose based medium than other, and this was true atdifferent levels of ISe. Among rest of the media, themaximum sugar consumption was recorded in jaggeryfollowed by molasses and APE. Similar consumptionpattern was observed for total sugar. The difference insugar consumption of the media from that of glucosemedium would be attributed to the presence of someunfermentable sugars in these media. The pH valuewas found to be the lowest in APE medium (4.40),followed by molasses, jaggery and glucose. It is inconsistence with the original pH values of therespective medium. The ethanol content recordedamong different media showed a significantly highermean production in glucose based medium (3.69%)than jaggery (3.20%), molasses (3.04%) and APE(2.83%), irrespective of ISe. It corroborated with thepattern of sugar consumption.

The average CYC was found to be significantlyhigher in glucose medium (0.28 gig sugar consumed).The rest of the media were, however, statistically at

223

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par with each other. The lowest CYC (0.23 gig sugarconsumed) was found in molasses medium. Further,the trend (Fig. 2) showed that maximum sugarconsumed during the growth by yeast was in glucosebased medium which produced more ethanol andbiomass than all other media. The reason can possiblybe traced to the presence of glucose as the onlyfermentable substrate, which is readily metabolizedby the yeast into ethanol and CO2 besides for its owngrowth. The presence of type of fermentable sugar inthe medium was also reported to effect theirconsumption by the yeast (aura, 1974). The ethanolcontent increased simultaneously as per the sugarconsumption pattern in respective media, beinghighest in glucose medium and lowest in APE. As asimple batch type anaerobic fermentation processwithout shaking, Saccharomyces cerevisiaeexpectedly produced ethanol and CO2 in all the mediatried (Amerine et al, 1980; Joshi, 1998b). The trendfurther disclosed that glucose medium gave higherbiomass yield than other media as evident from CYC(0.28 gig sugar consumed). Molasses mediumproduced the lowest CYC and this decrease might bedue to the presence of some growth inhibitorysubstances in the medium (Reed, 1982). Earlier, asubstrate yield of 7.5-10.5 g cell mass per 100 g offermentable sugar under anaerobic condition wasobtained (White, 1954; Reed, 1982). Under aerobicfed-batch conditions, a CYC of 0.407-0.55 gig sugarhas been reported, using different media and growthstimulators. Therefore, the CYC (0.23-0.28 gig sugarconsumed) recorded in present study is quitecomparable and is within the range obtained in earlierfindings (aura, 1974, 1976; Aiba et ai, 1976). Thedeviation, might be attributed to the difference in typeof process used, extent of continuous shaking, typeand amount of fermentable sugar in the medium.

224 INDIAN J BIOTECHNOL, APRIL 2003

The ISC of 5%, irrespective of carbon source used,seems to be optimum in the medium. Among differentsubstrates, glucose medium significantly enhanced thebiomass yield. Also, the APE had supported higherbiomass yield though at par with jaggery, clearlyshowing its suitability for the production of baker'syeast. It also produced average lowest ethanolcontent, which is desirable. Overall, apple pomace hasproved itself a good substrate for baker's yeastproduction.

Effect of Different Growth StimulatorsA number of minerals and vitamins were tried

along with the addition of yeast extract, beef extract,peptone and nitrogen source (ammonium sulphate) assuggested earlier for higher biomass production(Harrison, 1967; Wang et al 1977; Olson & Johnson,1949). The results (Table 2) spell out that averagereducing sugar consumption has increasedsignificantly with the addition of minerals (L2),

vitamins (L3), minerals and vitamins both (L4) thanthe control (L,), irrespective of carbon source.Significantly highest mean sugar consumptionoccurred in L4 (3.91 %) though statistically at par withL3 (3.83%) when compared with L, (3.67%). Thisimplies that the growth stimulators have exerted asignificant effect on baker's yeast fermentationbehaviour. The pH values of the media were alsoaffected by the addition of growth stimulators,especially minerals (L2). The higher average pH value(4.70) was recorded in control treatment (L,) than L2'L3 and L4. The subsequent decrease in the pH valuemight be attributed to the presence of organic and/orinorganic acids III growth stimulators. The ethanolcontents increased with the addition of growthsupplements as compared to the control treatment,irrespective of carbon sources. A significantly higherethanol contents were recorded in L4 (1.33%) and L3(1.27%) than L, and L2. It is inferred that the additionof growth stimulators increased, to some extent, theefficiency of yeast to ferment the sugar III therespective media. The CYC was also enhanced by thesupplementation of media with growth stimulators,irrespective of carbon sources. A significantly higherCYC was recorded in L4' L3 and L2 in comparison toL, (0.28 gig sugar). The results clearly reflect thatgrowth stimulators had significant effect on thebiomass production of baker's yeast, irrespective ofcarbon source employed.

As the type of growth stimulators were changedfrom L, to L4, subsequent increase III CYC wasobtained with only a slight increase in ethanol

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production and sugar consumption (Fig. 1). Thus,addition of growth stimulators increased theefficiency of baker's yeast to respire and/or fermentthe fermentable sugar present in the respective media.It is also in confirmation with the earlier findings onglucose and molasses media, where addition of suchgrowth stimulators increased the efficiency of yeastfor higher biomass production (Frey et al, 1936;White, 1954; Harrison, 1967; Wang et al, 1977; Joshiet al 1998; Olson & Johnson, 1949; Reed, 1982).

The changes that occurred during fermentationusing different carbon sources, irrespective of growthstimulators (Fig. 2 ) clearly show the superiority ofglucose with respect to higher CYC and ethanolproduction over rest of the substrates. The same trendin sugar consumption was also exhibited. AmongAPE, molasses and jaggery medium, APE gave higherCYC with lower ethanol production as per the amountof sugar consumed. Thus, APE is second to glucose inthe medium which supported the higher yeastbiomass. The CYC (0.35 gig sugar consumed)obtained in this experiment was lower than 0.48-0.56gig sugar under aerobic, fed-batch culture (Wang etal, 1977, 1979; aura, 1974, 1976; Aiba et al, 1976),but higher than 0.075-0.11 gig sugar consumed underanaerobic conditions (White, 1954; Reed, 1982). Theinteraction of growth stimulators and carbon sourcefurther revealed (Table 2) that sugar consumption inglucose medium was increased with the addition ofgrowth stimulators than control. But in case of theAPE, baker's yeast fermentation behaved differently.The sugar consumption increased but non-significantly with the addition of growth stimulatorsas compared to control (L,). It shows that there is nosignificant effect of addition of either minerals andvitamins (L3) alone or in combination (L4). It might bedue to the occurrence of various growth supportingnutrients in apple pomace (Smock & Neubert, 1950;Hang et al, 1989; Kaushal, 1992; Kennedy, 1994;Devarajan, 1997; Joshi, 1998a). However, molassesand jaggery exhibited similar pattern of sugarconsumption to that of glucose. The ethanol contentsand CYC also increased (as per the increase in sugarconsumption between different media) with theaddition of growth stimulators alone (L2 and L3) or incombination (L4) as compared to control treatment(L,). APE medium showed a non-significant increasewith respect to ethanol content and CYe. Thefermentation behaviour suggested thatsupplementation of the APE medium with variousgrowth factors for the production of baker's yeast has

225

no significant effect and thus, such addition is notrequired except the normal growth supplements suchas yeast extract, beef extract, and ammonium sulphate(which were common in all the media tried).However, most of the reports on the requirements ofbaker's yeast for various minerals and vitamins wereusually based on the concentration of these growthstimulators in the growth media (Olson & Johnson,1949; Von Meyenbarg, 1969; aura, 1974; Wang et al,1977), but no correlation with the biomass yield hasbeen mentioned to give the amount of yeast produced.

ConclusionGlucose was found the best energy source for

production of baker's yeast, followed by APE. Sincethe addition of growth stimulators did not affect thesugar consumption, ethanol production andlor CYC,there is no need to add either mineral or vitamins tothe medium while using APE. The presence of higherpercentage of fermentable sugar per unit totalcarbohydrate, higher crude proteins, absence ofgrowth inhibitory compounds as known to be presentin molasses, qualify APE to be a good substrate forbaker's yeast production.

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