Influence of adjuncts as a debittering aids in encountering the bitterness developed in cheese...

Original article

Influence of adjuncts as a debittering aids in encountering the

bitterness developed in cheese slurry during accelerated ripening

Kumar Sudhir,1* Yogesh Kumar Jha2 & Singh Pratibha3

1 Department of Food Science and Dairy Technology ⁄Biotechnology, Baba Farid Institute of Technology, Dehra Dun-248001 (Uttarakhand),

India

2 Department of Food Science and Technology, G.B. Pant University of Agriculture and Technology, Pantnagar-263 145 (Uttarakhand), India

3 SMS, KVK, Dhakrani, Dehra Dun (Uttarakhand), India

(Received 19 August 2009; Accepted in revised form 31 March 2010)

Summary An attempt was made to accelerate the flavour development in cheese base with the help of exogenous

proteolytic and lipolytic enzymes (1:1 proportion, each at the rate of 0.025% by weight of cheese-base) and

ripening at elevated temperatures (i.e. 20 ± 1 �C) for up to 12 days. To counter the bitterness developed,

adjunct cultures were used: viable or attenuated (freeze-shocked or heat shocked). Study of biochemical

characteristics, electrophoretic pattern and sensory evaluation of the product were carried out. An acceptable

enzyme-modified, lightly salted cheese base was obtained using 0.025% each of proteolytic and lipolytic

enzymes, along with 5% starter culture and adjuncts followed by ripening up to 12 days. Freeze-shocked

adjunct Lactobacillus helveticus produced enzyme-modified cheese base with no detectable bitterness. The

usage of exogenous enzymes, temperature of ripening, ripening period and interactions amongst these

parameters had significant (P < 0.01) influence on all of the biochemical characteristics monitored.

Keywords Accelerated cheese ripening, adjuncts, bitterness, debittering potential, electrophoretic patterns, Lactobacillus helveticus.

Introduction

Cheese, the nature’s wonder food and the classicalproduct of biotechnology, is a highly nutritious foodwith good keeping quality, enriched pre-digested foodwith fat, calcium, phosphorous, riboflavin and othervitamins, available in a concentrated form. Besides, it isthe only concentrated and balanced milk food forlactose intolerants. The supremacy of cheese technologystrongly lies in the fact that it not only retains theoriginal biological value of milk, but further enhances itby virtue of its bio-available beneficial microflora andprobiotics. Cheese has become a major food product inEurope, North America and South America and NewZealand. The annual growth rate is reported to be10–15% and the price realisation is higher in cheesecompared to traditional dairy products (Fox & Tobin,2005). In cheese ripening, milk proteins undergo con-trolled proteolysis under the combined influence ofnative micro flora, starter bacteria and rennet. Ripeningof cheese is a very complex and in many cases a slowbiochemical process. Numerous biochemical and phys-ical changes can occur in cheese curd during distribution

and storage. Cheese flavour is one of the most importantcriteria determining consumer choice and acceptance.Cheese flavour varies widely with source, age and fatcontent. It takes 4–12 months to develop full cheesyflavour in Cheddar cheese. Flavour in cheese resultsfrom interplay of the fat and protein components andthe products of the break down. The biochemicalchanges in the curd matrix responsible for the develop-ment of the cheesy flavour are the metabolism ofresidual lactose, lactate and citrate (sometimes, althougherroneously, referred to as ‘glycolysis’), liberation of freefatty acids, FFA (lipolysis), associated catabolic reac-tions and the degradation of the casein matrix to a rangeof peptides and free amino acids, FAA (proteolysis),and subsequent reactions involved in the catabolism ofFAA (McSweeney & Sousa, 2000). The primary reac-tions of glycolysis, proteolysis and lipolysis form prod-ucts that then undergo numerous changes, whichdetermine the characteristics flavour and texture of eachcheese variety.Owing to the cost of inventory and controlled

atmosphere ripening rooms, the loss of cheese massthrough evaporation and the risk that the quality of themature cheese may be far from satisfactory, cheeseripening is a relatively expensive process. The dairy*Correspondent: E-mail: [email protected]

International Journal of Food Science and Technology 2010, 45, 1403–1409 1403

doi:10.1111/j.1365-2621.2010.02280.x

� 2010 The Authors. Journal compilation � 2010 Institute of Food Science and Technology

industry is faced with the challenge of acceleratingcheese ripening (ACR) with a flavour and texture asattractive as those in a fully ripened cheese. There areeconomic and technological incentives to accelerate therate of ripening and reduce costs. A controlled methodof accelerated cheese ripening, achieved without upset-ting the flavour balance, would give large savings.Addition of curd slurries, starter adjuncts, microbialrennets, exogenous enzymes viz., proteases and lipasessingly or in combination, microencapsulated enzymespreparations and supplementation of buffalo milk withgoat milk helped in accelerated ripening of cheese (Singh& Kanawjia, 1993; Fox et al., 1996). Chapot-Chartieret al. (1994) reported that cheese made with Lactococcuslactis subsp. cremoris AM2 developed a higher level ofamino nitrogen and was less bitter than that made withstrain Lactococcus lactis subsp. cremoris NCDC 763.Crow et al. (1995) induced early lysis of Lactococcus bya low level of phage infection without any impairment ofacid production. They reported that phage treatmentaccelerated the decline in viable starter numbers duringripening, accelerated the release of free amino acids andammonia and reduced bitterness. Martinez-Cuesta et al.(1998) studied the effect of the bacteriocin producerLactococcus lactis IFPL 105, which is able to induce thelysis of starter adjuncts with high peptidase activity,Lactobacillus casei subspecies casei IFPL 731 andLactococcus lactis subspecies lactis T1 on proteolysisin cheese curd slurries. They reported that the inclusionof a bacteriocin producer as an adjuncts gave promisingresults. It did not interfere with acidification of the curdby the starter and resulted in increased levels of aminonitrogen, which is correlated, with the early lysis ofadjuncts. Drake et al. (1996) reported that addition ofLactobacillus helveticus WSU19 to milk at a level of 102–103 cfu mL)1 accelerated ripening and intensified theflavour; the adjunct increased the nutty flavour andimproved flavour acceptability. Cheevers et al. (1996)found similar effect for Lactobacillus helveticus DPC4571, which dies quickly in the cheese and autolyses.Adjuncts containing cheeses underwent acceleratedproteolysis and received higher sensory scores than thecontrols.Cheese flavour is a complex mixture of several

hundred flavour components. The final flavour in cheeseis achieved during the prolonged ripening process whena bland, elastic curd is transformed into a well bodiedcheese (El-Soda & Pandian, 1991). It is relatively easy toaccelerate many or most of the reactions involved incheese ripening but it is much more difficult to acceleratethe complex set of reactions in a balanced manner as tomaintain or perhaps to improve cheese quality. Sinceripening is expensive and to some extent, unpredictable,acceleration of ripening is desirable provided thatproper balance is maintained. Usage of adjuncts culturesis one such technique. Adjunct culture is a new

technology used by the cheese industry to offer to theconsumers’ safe and consistent cheese with high orga-noleptic properties in a reasonable ripening time.Adjunct cultures can be defined as selected strains ofcheese related microorganisms that are added to thecheese milk ⁄ cheese curd to improve developmentof cheese sensory quality. They were also developed toaccelerate cheese ripening, which may allow substantialcost savings to the cheese industry.The present investigation envisaged standardisation

of a procedure to accelerate cheese ripening processusing an integrated approach. Attempts to improve theflavour attributes of cheese are dictated first andforemost by the view that such a complex, delicatelybalanced process can be influenced without adverseeffects only by increasing the proteolytic potential in thecheese in all aspects of its specificity with respect to thedegradation of casein. Moreover, a simultaneousincrease of enzyme systems of the starter and adjunctsthat possibly are responsible for the conversion ofproducts of proteolysis and lipolysis to flavour compo-nents should not be disregarded. This work aimed toassess the role of adjuncts as debittering aids inremoving the bitterness during accelerated cheeseripening.

Materials and methods

Milk

Buffalo milk was obtained from Instructional DairyFarm of the University. The proximate composition ofbuffalo milk standardised to casein ⁄ fat ratio of 0.70 was84.36% moisture and 15.64% total solids; 3.52%protein, 6.90% fat, 0.83% ash and 4.39% carbohydrates(by difference).

Starter culture

LF-40, a mixed culture comprising Lactococcus lactisvar. diacetilactis, Lc. lactis subsp. lactis and Leuconostocsp. was used as starter culture. Freeze dried ampules ofthe cultures were obtained from National Collection ofDairy Cultures, Dairy Microbiology Division, NDRI,Karnal. It was then propagated in sterilised litmus milk.The freeze dried culture was transferred aseptically to atest tube containing 10 mL sterilised litmus milk andincubated at 22 ± 1 �C until the curd set. The activityof the culture was maintained by daily transfer. Steri-lised buffalo skim milk was used for the preparation ofbulk culture.

Exogenous enzymes

Promod was supplied by Bio-catalysts (Pontypridd,UK). It is a blend of 1:1 leucine amino peptidase to

Influence of adjuncts in encountering the bitterness developed in cheese slurry K. Sudhir et al.1404

International Journal of Food Science and Technology 2010 � 2010 The Authors. Journal compilation � 2010 Institute of Food Science and Technology

endo-proteinase derived from mixed strains of Aspergil-lus sp. Lipomod enzyme was supplied by Bio-catalysts.This enzyme is specially formulated mixed fungal lipasedesigned to promote blue cheese flavour. These enzymeswere dissolved in 5 mL of distilled, sterile saline water.

Preparation of cheese base

The method recommended by Rajak & Jha (2000) wasused to prepare cheese base. Buffalo milk standardisedto a casein to fat ratio of 0.70 (fat 6.9%), waspasteurised at 90 ± 1 �C for 4–5 min and cooled to60 ± 1 �C. Milk was coagulated with 2% citric acidsolution and the curd separated using muslin cloth; 21%was the yield of cheese curd.

Preparation of cheese slurry

Curd slurry was prepared by mixing two parts of freshcurd with one part of sterile distilled water in a powerblender (Model Fx10, Wattage 600 W, rpm 18 000) for3–4 min, common salt at 2% and, starter culture wereadded to the enzyme blends at different rates. Stricthygienic conditions were observed. Utensils were steri-lised with hot water. The slurries were transferred intofresh, clean, sterilised jars (UV treated, UV 1 · 30 W,distance 21 in.). Ladle used for mixing was sterilisedwith 70% alcohol. Mixing of the slurries was done in alaminar airflow chamber (Model C-42, Air-1 · 1 ⁄6HP,3speed, Max. 1375RPM, light-2C40W, UV 1 · 30W)manufactured by CleanAir Scientific Aids, New Delhi,India.The moisture content of the cheese slurry was

determined by AOAC (1975) procedure. The ash con-tent of the cheese slurry was determined as per theprocedure outlined in AOAC (1975). The nitrogen wasdetermined by micro-Kjeldahl method as per the pro-cedure outlined in AOAC (1975) and the protein contentwas calculated using a conversion factor of 6.38. The fatcontent of the cheese slurry was estimated by SoxhletExtraction method (AOAC, 1975).The proximate composition of cheese slurry was

moisture 61.2%, protein 18.5%, fat 15.5% and ash1.5%.

Analysis of cheese slurry sample during ripening

Biochemical characteristics were assessed at an intervalof 0, 4, 8 and 12 days. pH of the cheese wasdetermined by the method of AOAC (1975) using adigital pH meter (Electronic Corporation of India Ltd,Hyderabad, India, type 101), titratable acidity (as %lactic acid) by the method of AOAC (1975), solublenitrogen by the method of Kosikowski (1970) and freefatty acid (FFA) by the method of Deeth & Fitz-Gerald (1976).

SDS-PAGE

Electrophoresis was done by the method of Laemmli(1970).Electrophoresis was done at a constant current of40 mA per gel and a voltage of 121 V. Gels were run forabout 4 h. The cheese curd protein (200 mg) wasextracted by the method of Bhowmik et al. (1990) forthe purpose of SDS-PAGE. An aliquot of 10 lL wasused for loading.

Attenuation of Adjunct cultures

This was done as per the method given by Ezzat & El-Shafei (1991).The cultures were sub-cultured into Elliker broth 2–3

times. The broth was centrifuged at 10,000 · g (4 �C)for 5 min (Plastocrafts, Mumbai, India) when theculture reached the stationary phase. The cell pelletwas washed thrice with 0.01 M phosphate buffer (pH7.0) and finally suspended in sterile peptone water. Thecell suspension was frozen for 24 h at )20 �C, afterwhich it was thawed in water at 40 �C. The suspensionwas centrifuged at 10,000 · g (4 �C) for 5 min (Plasto-crafts) and the cell pellet was suspended into sterile skimmilk. This was then used as freeze-shocked adjunctculture. Viable cell counts were made of the cellsuspension after growth and thawing of the culture, onElliker broth. Untreated whole cells of Lactobacilluscasei and Lactobacillus helveticus were from the samefermentation as freeze-shocked cells, but were stored insterile skim milk at 4 �C overnight after centrifugationand washing procedure.The cultures were sub-cultured into Elliker broth two

to three times. The broth was centrifuged at 10 000 · g(4 �C) for 5 min (Plastocrafts) when the culture reachedthe stationary phase. The cell pellet was washed thricewith 0.01 m phosphate buffer (pH 7.0) and finallysuspended in sterile peptone water. The cell suspensionwas heat-shocked at 67 �C (L.casei for 22 s andL.helveticus for 15 s), after which it was kept at roomtemperature. The suspension was centrifuged at10 000 · g (4 �C) for 5 min (Plastocrafts) and the cellpellet was suspended into sterile skim milk. This wasthen used as heat-shocked adjunct culture. Viable cellcounts were made of the cell suspension after growth ofthe culture, on Elliker broth. Untreated whole cells ofLactobacillus casei and Lactobacillus helveticus werefrom the same fermentation as freeze-shocked cells, butwere stored in sterile skim milk at 4 �C overnight aftercentrifugation and washing procedure.

Sensory evaluation

Sensory characteristics and overall acceptability ofcheese slurry were assessed by a panel of nine trainedpanellists consisting of post-graduate students and

Influence of adjuncts in encountering the bitterness developed in cheese slurry K. Sudhir et al. 1405

� 2010 The Authors. Journal compilation � 2010 Institute of Food Science and Technology International Journal of Food Science and Technology 2010

faculty members of the Department of Food Scienceand Technology, College of Agriculture, Govind Bal-labh Pant University of Agriculture and Technology, onthe basis of 9-point hedonic scale (1 = dislike extre-mely; 5 = neither like nor dislike and 9 = like extre-mely). Sensory characteristics studied included flavour,body and texture, appearance and overall acceptability.The scores were given in the decreasing order with 9 for‘like extremely’ and 1 for ‘dislike extremely’. In spite oftrained sensory panels, they were exposed to character-istics cheese flavour by giving standard cheese slurrysamples at least for 30 days at an interval of 3–4 days.Cheese slurry samples having sensory scores below 7were not acceptable and were rejected.Sensory evaluations were carried out after 0, 4, 8 and

12 days of ripening at 20 �C. These sampling periodswere established in tune with the production of flavourcompounds and the biochemical reactions occurring inthe cheese slurry. Samples of 25 g were presented to thepanellists monadically and evaluated on a nine pointHedonic scale. Samples were randomly assigned andorder of presentation was balanced among panellists.The panellists evaluated all three replicates for eachtreatment.

Statistical analysis

The experimental data obtained for biochemical andsensory characteristics were statistically analysed foranalysis of variance as described by Snedecor &Cochran (1967). Each experiment was replicatedthrice. The software spss for Windows (SPSS Inc.,

Chicago, IL, USA, version 8.0) was used in allstatistical procedures.

Results and discussion

The changes in glycolysis dynamics, measured in termsof pH and titratable acidity, due to adjunct actionduring ripening of cheese slurry samples are shown inTables 1 and 2. The pH of all cheese slurry samplestreated with adjunct cultures exhibited a linear decreas-ing trend. As compared to control samples made withonly starter culture LF-40, the final pH of cheese slurrysamples made with adjunct lactobacilli was much lower.Addition of viable adjunct lactobacilli resulted indecrease in pH but with attenuated adjuncts, the fallin pH was checked. Among attenuated adjuncts, mini-mum pH (4.32) was observed with freeze-shockedLactobacilllus helveticus at 12 days ripening. Regardingattenuation methods, freeze-shocking had more impactthan heat-shocking in reducing the pH less. This may bepartially due to the thermal denaturation of the auto-lytic system of the cultures. Reverse trend was observedin titratable acidity. The per cent titratable acidityincreased linearly as the ageing time progressed. Thetitratable acidity in adjunct treated cheese slurry wasmore in comparison to cheese slurry with only starterculture LF-40. Maximum titratable acidity was observed(1.44% lactic acid) was observed with viable adjunctLactobacillus helveticus at 12 days ripening. With atten-uated adjuncts, maximum titratable acidity (1.38%lactic acid) was observed with heat-shocked Lactobacil-lus helveticus at 12 days ripening. The impact of

Table 1 Effect of adjunct Lactobacillus casei on biochemical characteristics of cheese slurry during ripening at 20 �C

Parameters ⁄treatments

Nature of

adjunct

Days of ripening

0 (6 hr.) 4 8 12

A B C D A B C D A B C D A B C D

pH V 5.42 5.38 5.31 5.45 5.14 5.13 5.10 5.12 4.88 4.81 4.78 4.96 4.51 4.41 4.37 4.73

HS 5.40 5.40 5.41 5.46 5.24 5.21 5.21 5.11 4.81 4.71 4.70 4.96 4.63 4.60 4.54 4.73

FS 5.41 5.41 5.39 5.45 5.20 5.24 5.18 5.12 4.80 4.76 4.75 4.95 4.60 4.58 4.51 4.76

TA V 0.31 0.43 0.57 0.24 0.72 0.99 1.13 0.44 0.98 1.23 1.29 0.63 1.09 1.32 1.38 0.69

HS 0.30 0.41 0.54 0.23 0.69 0.95 1.09 0.44 0.94 1.18 1.24 0.61 1.05 1.27 1.33 0.69

FS 0.28 0.40 0.52 0.23 0.66 0.91 1.04 0.43 0.90 1.13 1.19 0.61 1.0 1.21 1.27 0.68

Soluble protein % V 1.30 1.58 1.51 0.88 2.52 3.24 3.16 1.84 4.04 5.44 5.02 3.15 6.39 7.38 7.07 4.51

HS 1.35 1.38 1.42 1.10 2.82 3.68 3.56 1.96 4.67 6.13 5.57 3.16 7.21 8.43 7.92 4.49

FS 1.34 1.63 1.58 1.13 3.09 3.91 3.79 2.10 5.02 6.60 5.99 3.21 7.92 9.05 8.52 4.60

FFA (% oleic acid) V 1.23 1.46 1.40 0.87 2.18 3.38 3.24 2.01 4.48 5.45 5.22 3.21 5.91 6.44 6.28 4.52

HS 1.13 1.34 1.28 1.10 2.50 3.11 2.98 1.97 4.12 5.01 4.80 3.15 5.44 5.92 5.78 4.51

FS 1.18 1.40 1.34 1.12 2.66 3.21 3.08 1.99 4.05 5.18 4.96 3.22 5.62 6.12 5.97 4.62

Where A = 0.025% each of Lipomod and Promod +2% adjuncts +5% LF 40; B = 0.025% each of Lipomod and Promod +2.5% adjuncts +5% LF 40;

C = 0.025% each of Lipomod and Promod +3% adjuncts +5% LF 40; D = 0.025% each of Lipomod and Promod +0% adjuncts +5% LF 40 (control).

Each value is mean of three independent determinations (the SD between two values = 0.01).

V, viable adjunct; HS, heat shocked; FS, freeze shocked.



Lactobacillus helveticus was more than the effect ofLactobacillus casei. Among the different levels ofadjunct used, 2.5% of adjunct gave the most promisingresults. The effect of various treatments viz., nature andtype of adjunct cultures, amount of adjunct cultureadded and ripening period and interactions among theseparameters significantly influenced the changes in titrat-able acidity (P < 0.01) (Table 3). The addition of viableadjunct lactobacilli resulted in additional lactic acidproduction, causing a significant decrease in the finalpH. Attenuation probably resulted in drastic suppres-sion of lactic acid synthesis or lactose metabolism. Thismay be partially attributed to the thermal denaturationof the autolytic system of the adjuncts.The changes in soluble protein of cheese slurry

samples due to adjuncts incorporation are depicted in

Tables 1 and 2. The level of soluble protein is anindicator of the proteolytic activity of the adjunctcultures. The protein degradation in the study is clearlydemonstrated by the definitive increase in solubleprotein concentration of all treated cheese, whichincreased (P < 0.01) as ripening time progressed. Theproteolysis in adjuncts treated cheese slurry samples washigher in comparison to control cheese slurry samples.Among adjuncts, attenuated adjuncts showed higherproteolysis than viable adjuncts. Among attenuatedadjuncts, freeze-shocked adjuncts were more effective inenhancing proteolysis than heat-shocked adjuncts.Between the two adjuncts, Lactobacillus helveticus hada more pronounced effect than Lactobacillus casei. Theeffect of various treatments viz., nature and type ofadjunct cultures, amount of adjunct culture added and

Table 2 Effect of adjunct Lactobacillus helveticus on biochemical characteristics of cheese slurry during ripening at 20 �C

Parameters ⁄treatments

Nature of

adjunct

Days of ripening

0 (6hr.) 4 8 12

A B C D A B C D A B C D A B C D

pH V 5.40 5.35 5.28 5.44 5.18 5.21 5.23 5.10 4.74 4.75 4.70 4.94 4.48 4.38 4.32 4.75

HS 5.41 5.37 5.34 5.43 5.21 5.30 5.24 5.10 4.78 4.81 4.76 4.94 4.58 4.54 4.51 4.76

FS 5.40 5.38 5.32 5.43 5.21 5.28 5.26 5.10 4.84 4.80 4.78 4.94 4.61 4.56 4.48 4.75

TA V 0.35 0.47 0.61 0.23 0.86 1.17 1.25 0.45 1.09 1.36 1.43 0.61 1.21 1.39 1.44 0.68

HS 0.34 0.41 0.59 0.23 0.83 1.12 1.20 0.42 1.04 1.31 1.37 0.62 1.16 1.33 1.38 0.67

FS 0.31 0.43 0.56 0.24 0.79 1.08 1.15 0.45 1.00 1.25 1.29 0.60 1.11 1.28 1.33 0.71

Soluble protein % V 1.39 1.64 1.62 0.93 2.71 3.49 3.40 1.93 4.45 5.86 5.34 3.22 6.95 7.94 7.61 4.62

HS 1.38 1.40 1.43 1.12 3.03 3.95 3.80 1.94 4.98 6.54 5.86 3.18 7.76 8.86 8.34 4.55

FS 1.41 1.65 1.64 1.11 3.26 4.16 4.08 1.95 5.35 7.03 6.30 3.28 8.34 9.53 8.97 4.65

FFA (% oleic acid) V 1.16 1.37 1.30 0.89 2.64 3.17 3.04 2.09 4.26 5.17 4.80 3.25 5.32 5.86 5.90 4.49

HS 1.07 1.26 1.20 1.11 2.42 2.92 2.80 1.95 3.92 4.76 4.42 3.20 4.89 5.39 5.43 4.52

FS 1.11 1.32 1.25 1.10 2.50 3.01 2.89 1.93 4.05 4.91 4.56 3.24 5.05 5.57 5.61 4.64

Where A = 0.025% each of Lipomod and Promod +2% adjuncts +5% LF 40; B = 0.025% each of Lipomod and Promod +2.5% adjuncts +5% LF 40;

C = 0.025% each of Lipomod and Promod +3% adjuncts +5% LF 40; D = 0.025% each of Lipomod and Promod +0% adjuncts +5% LF 40 (control).

Each value is mean of three independent determinations (the SD between two values = 0.01).

V, viable adjunct; HS, heat shocked, FS, freeze shocked.

Table 3 anova of biochemical changes with respect to adjunct type, its levels and time of ripening

Source of variation d.f.

F-value C.D. at 5% level

pH

Titratable

acidity

Soluble

protein

Free fatty

acid pH

Titratable

acidity

Soluble

protein

Free fatty

acid

Type of adjunct cultures (A) 5 4.815 32.190 1979.371 664.175 0.035 0.022 0.019 0.022

Amount of adjunct cultures (B) 3 1261.094 1744.616 34182.16 79163.99 0.029 0.018 0.016 0.018

Ripening period (C) 3 17.396 2618.029 197313.5 5579.249 0.029 0.018 0.016 0.018

A*B 15 3.537 4.112 152.274 96.68 0.071 0.044 0.038 0.044

B*C 9 12.621 57.167 3693.339 402.46 0.071 0.044 0.038 0.044

A*C 15 2.380 1.999 254.658 637.702 0.058 0.036 0.031 0.036

A*B*C 45 1.808 0.612ns 40.456 71.439 0.141 0.087 0.077 0.088

All values are significant at 1% level unless mentioned otherwise.

ns, non-significant.



ripening period and interactions among these parame-ters significantly influenced the changes in solubleprotein (P < 0.01) as is evidenced from table 3. Withfreeze-shocked Lactobacillus casei and Lactobacillushelveticus, maximum soluble protein observed was 9.05and 9.53% respectively whereas with heat-shockedLactobacillus casei and Lactobacillus helveticus, it was8.43 and 8.86% respectively. Freeze-shockedLactobacillus helveticus caused a pronounced increasein the levels of soluble protein in comparison to freeze-shocked Lactobacillus casei. This may be due to the lysisof freeze-shocked Lactobacillus helveticus to a greaterextent than freeze-shocked Lactobacillus casei. It mightalso be attributed to the lower destruction of theproteolytic enzymes of the Lactobacillus helveticusduring freezing. These results are in agreement withthe findings of Aly (1990a,b) and Kebary et al. (1996).El-Soda et al. (1991) reported a 52% increase inproteolysis (viz., trichloroacetic acid soluble nitrogen)in 6 months old cheese made with frozen Lactobacilluscasei cells. The higher rate of proteolysis may beattributed to increased action of cell wall proteinases.Lactobacillus helveticus is known to possess a propyl-dipeptidylase enzyme that can further hydrolyse thepeptides produced. This aminopeptidase has contrib-uted for development of cheese flavour by producingfree amino acids during ripening (Khalid et al., 1991).The increase in the soluble protein content of cheeseslurry samples conclusively proves the enhanced prote-olytic activity of the attenuated adjunct lactobacilli. Ourresults are in line with the findings of Ardo & Mansson(1989).They reported that in cheese samples containingheat-shocked lactobacillus, casein was not degradedfaster, although peptidolysis proceeded at a faster rate.Our results are also in agreement with the findings ofTungjaroenchai et al. (2001). They reported that cheesecontaining adjunct Lactobacillus helveticus underwentaccelerated proteolysis than the control cheese. Degra-dation patterns of cheese proteins and casein moieties byelectrophoresis analysis (Plate 1–2) revealed that differ-ence existed in proteolytic pattern of the protein in thecheese slurry samples. SDS-PAGE characteristics ofcasein moieties of differently treated cheese slurryshowed that the initial intensity of b-casein band wasmuch greater than the as1-casein, casein bands graduallybecome weaker as the ageing progressed. The b-caseinbond remained somewhat intact in control cheese andwas degraded differently in adjunct treated cheese slurrysamples. The initial cheese (zero day) showed that as1-casein was the major protein bands in cheese slurry. Theas1-casein gradually disappeared on 6th day. Theintensities of c1-casein and small peptides increased upto 12th day. The disappearance of b-casein in adjuncttreated cheese slurry was correlated with increase insoluble protein content (Tables 1 and 2). The b-caseinhas been implicated for bitterness in cheese slurry

samples and its degradation and hydrolysis led toincrease in flavour score without any detectable bitter-ness.Lipolysis in control and experimental cheeses during

ripening as indicated by total free fatty acids (measuredas % oleic acid) is shown in Tables 1 and 2. Fatdegradation in this study is clearly demonstrated by thedefinitive increase in free fatty acid concentration of alltreated cheese, which increased (P < 0.01) as ripeningtime increased. The adjunct treated cheese in generalexhibited significantly higher (P < 0.01) levels of FFAliberation with the progress of ripening as comparedwith control cheese. Among adjuncts, Lactobacillushelveticus had a more pronounced effect than Lactoba-cillus casei. Between different levels 2.5% of adjunctgave the best result. Cheese made with freeze-shockedLactobacillus casei formed the highest level of FFA(6.12) compared with all other adjunct treated cheeses.The effect of various treatments viz., nature and type ofadjunct cultures (A), amount of adjunct culture added(B) and ripening period (C) and interactions amongthese parameters significantly influenced the changes inlipolysis at P < 0.01 (Table 3). These results indicatethat adjunct lactobacilli contribute to lipolysis in cheeseslurry and different adjunct strains have different lipo-lytic activity.Enhanced rate of lipolysis with added adjunct during

cheese ripening may be due to liberation of intracellularlipase upon lysis and this may account for the enhancedlipolytic activity in cheese slurry incorporated withlactobacilli adjuncts. Certain strains of lactobacilli showhigh activity of intracellular lipase upon autolysis, whichmay account for the higher lipolysis during ripening ofsame type of cheese. Khalid & Marth (1990) alsoreported similar findings. The findings of the presentinvestigation are also in agreement with those reportedby Aly (1990a,b), Ezzat & El- Shafei (1991) and Kebaryet al. (1996).Several workers have reported the enhancement of

glycotytic, lipolytic and proteolytic aspect of cheeseripening by attenuated lactobacilli. Increased values forripening indices viz., soluble nitrogen, PTA-SN, solubletyrosine, soluble tryptophan have been reported by Aly(1990a,b, 1994) and Kebary et al. (1996). They have alsoreported an increase in lipolysis in such cheeses.

Conclusions

In the present study, a process has been optimised todevelop enzyme modified cheese slurry with character-istic cheese flavour within a period of 12 days. Tostandardised this protocol, an integrated approach wasfollowed which implies usage of combination of cheeseslurry system, incorporation of exogenous enzymes,elevated ripening temperatures (20 ± 1 �C), attenuatedadjuncts lactobacilli in synergism. Incorporation of



exogenous enzymes at the level of 0.025% each ofLipomod and Promod with attenuated adjuncts (Freeze-shocked Lactobacillus helveticus) at the rate of 2.5% andstarter culture LF-40 (at the rate of 5.0%) in cheeseslurry, accelerated ripening in a balanced manner thatresulted in an enzyme modified cheese base with typicalcheese flavour.

References

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