Biochemical Engineering Journal 19 (2004) 181–187

Short communication

Production of multienzymes consisting of alkaline amylaseand cellulase by mixed alkalophilic culture and theirpotential use in the saccharification of sweet potato

Chong Zhang, Xin-Hui Xing∗, Min-Sheng LiuDepartment of Chemical Engineering, Tsinghua University, Beijing 100084, PR China

Received 24 September 2003; accepted after revision 1 July 2004

Abstract

Multienzymes of alkaline amylase and cellulase were produced by a mixed culture system consisting of the respective producers ofalkalophilicBacillus sp. The mixed culture enabled the two kinds of alkalophiles to grow in the same culture. Compared with the respectivepure cultures, the mixed culture exhibited a similar productivity of alkaline amylase, enhanced the secretion of alkaline cellulase from cellsurface to the culture broth, and lowered the protease activity that might be harmful to the other enzymes in practical application. Thesefindings show that the mixed alkalophilic culture is a useful method for the production of alkaline multienzymes. The crude multienzymesrecovered by ethanol precipitation exhibited well-designable activities of amylase and cellulase, which simultaneously degraded both thestarch and soluble cellulose.© 2004 Elsevier B.V. All rights reserved.

Keywords: Alkaline amylase; Alkaline cellulase; Enzyme production; Mixed microbial culture; Multienzymes

1. Introduction

Typical examples of natural mixed microbial culturesare activated sludge, anaerobic methane fermentation,and cheese production[1]. Biological processes using acontrolled/designed mixed microbial culture system haveexhibited high potential for the production of useful bio-substances compared with the conventional pure culture.Recently there have been reports of Vitamin B12 productionby a mixed culture consisting of two kinds of bacteria[2],PHB production by a periodically controlled mixed culture[3], kefiran production by a mixed culture ofLactobacilluskefiranofaciens and yeast[4], and production of industrialchemicals from cellulose by controlled mixed culture sys-tems[5]. The production of useful substances by couplingdifferent engineered microbes for the regeneration of ATPhas been developed successfully in Japan[6,7]. As impliedby these studies, a complex microbial culture system orwell controlled/designed mixed microbial culture system,by which microbial consortia are integrated in terms offunctionality and controllability, are promising methods forthe clean production of enzymes, fine chemicals, bioenergy,

∗ Corresponding author. Tel.:+86-10-6279-4771;fax: +86-10-6277-0304.E-mail address: xhxing@tsinghua.edu.cn (X.-H. Xing).

and a remedy for environmental pollution. As one applica-tion for the production of biosubstances by a designed mi-crobial consortium, this study selected a complex microbialsystem consisting of alkalophilic microorganisms capableof producing alkaline amylase and cellulase to producemultienzymes.

Multienzymes are defined as a system consisting of morethan two kinds of enzymes. They are expected to have someadvantages over single enzymes such as functional inte-gration and cooperative effect. Thus, multienzymes havehigh potential to many complex systems for processing,production, and POPs remediation. So far multienzymeshave demonstrated an important role in the production ofdetergent and food, and in the feed industry[8–10].

But currently there is still no appropriate bioprocesswith a well-established bioengineering basis to effectivelyand simultaneously produce multienzymes by a complexmicrobial culture system. Traditional ways to producemultienzymes include three methods. The first method in-volves the mixing of various enzymes directly, but thismethod is too costly. Another method uses the pure cul-ture of genetically-engineered microbes endowed withmulti-functions. This method depends on the characteristicsof host microbes and the genes to be introduced, but inmany cases multi-genes will be a heavy burden to host cells,which will limit the practical application. The third method

1369-703X/$ – see front matter © 2004 Elsevier B.V. All rights reserved.doi:10.1016/j.bej.2004.01.001

182 C. Zhang et al. / Biochemical Engineering Journal 19 (2004) 181–187

is a mixed culture consisting of different well-designed mi-crobes. This method is being paid much attention becausethe individual microbes in the mixed culture are easilymodified and the interaction between the microbes can beutilized in terms of the function integration for the effectiveproduction of bioproducts. Furthermore, the separation andpurification of multi-components can be achieved with onlyone culture. The bottle-neck problem for limiting the useof the mixed bacterial culture is the lack of quantitativeanalysis tool for the mixed system.

In the present paper, twoBacillus species producingextracellular alkaline amylase and cellulase[11,12] werechosen to construct a mixed alkalophilic culture system forproduction of multienzymes. Features of the microbial in-teraction for cell growth and enzyme production by mixedculture were examined by comparison with the respectivepure cultures. Furthermore, application of multienzymes insweet potato processing was also explored as a model.

2. Materials and methods

2.1. Bacterial strains

An alkaline amylase producer,Bacillus sp. JCM 9141, andan alkaline cellulase producer,Bacillus sp. JCM 9156, wereused as members of a mixed alkalophilic culture system inthis study. The two microbial strains can grow at pH 11, andboth of the alkaline enzymes are produced extracellularly[11,12].

2.2. Medium and culture conditions

Glucose medium: glucose, 10 g; trypton, 5 g; yeast extract,5 g; MgSO4, 0.2 g; K2HPO4, 1 g; Na2CO3, 10 g and 1 l ofdistilled water. Sodium carbonate was sterilized separatelyand added to the medium to adjust the pH at about 10.5.

Starch medium: starch, 20 g; trypton, 5 g; yeast extract,5 g; MgSO4, 0.2 g; K2HPO4, 1 g; Na2CO3, 10 g and 1 l ofdistilled water. Sodium carbonate was sterilized separatelyand added to the medium to adjust the pH at about 10.5.

CMC medium: CMC, 20 g; trypton, 10 g; yeast extract,5 g; NaNO3, 5 g; NaCl, 5 g; KH2PO4, 1 g; Na2CO3, 10 gand 1 l of distilled water. Sodium carbonate was sterilizedseparately and added to the medium to adjust the pH at about10.5.

For the alkaline amylase producer, a glucose medium anda starch medium were used for cultivation. For the alkalinecellulase producer, a glucose medium and a CMC mediumwere used for cultivation. For the mixed mixture, a glu-cose medium and a mixture of the starch medium and CMCmedium in a volume ratio of 1:1 (named mixed medium)were used.

The entire cultivation was carried out on a reciprocalshaker at 150 rpm and 37◦C. For the cultivation, a 500-mlErlenmeyer flask containing 100 ml of medium was inocu-

lated with a seedling culture of 1 day at an inoculum size of4% (v/v). In the mixed culture, each strain was inoculatedat an inoculum size of 2% (v/v).

2.3. Recovery of crude enzymes

After cultivation for a predetermined time, the cell-freeenzyme solutions were prepared by centrifugation at10,200× g for 10 min at 4◦C. Precipitation of the crudeenzymes was performed by addition of cold ethanol attwo-fold volumes, and subsequently by centrifugation at5700× g for 10 min at 4◦C. The recovered crude enzymeswere preserved in Tris–HCl buffer (pH 8.0).

2.4. Saccharification of sweet potato by recovered enzymes

One gram of dry sweet potato powder was added into100 ml water. Na2CO3 was added to adjust the pH to 11,and then the sample was heated to boiling, until the samplebecame a mash. Then a determined amount of amylase ormultienzymes was added to saccharify the mash at 40◦Cunder continuous agitation until the reducing sugar no longershowed any change.

2.5. Measurements

Cell concentration was measured by optical density at660 nm (OD660). The pH was measured with a compact pHmeter (Horiba Ltd., Japan).

2.5.1. Glucose concentrationOne ml of dinitrosalycilic acid solution was added to

1 ml sample solution. The mixture was heated in a boilingwater bath for 5 min, and then 4 ml of water were added.Absorbance of the sample was measured at 510 nm.

2.5.2. Assay of enzymatic activitiesThe enzymatic activities were measured according to

the procedures reported by Horikoshi and others[11,12].For measurement of alkaline protease activity, one ml ofcell-free sample solution suitably diluted was mixed with5 ml of 0.6% (w/v) Hammerstein casein solution (pH 11.5made up of 2× 10−2 M Na2HPO4–NaOH buffer) at 30◦C.After 10 min incubation, 5 ml of trichloroacetic acid (TCA)solution (consisting of 0.11 M TCA, 0.22 M sodium acetateand 0.33 M acetic acid) was added to the reaction mixture.The mixture was further incubated at 30◦C for 30 min andthen centrifuged at 10,200×g for 10 min; absorbance of thefiltrate was measured at 275 nm. One unit of the proteaseactivity was defined as the amount of the enzyme requiredto produce digestion which is not precipitated by TCA so-lution and which gives absorbance value to 1�g of tyrosineper min at 30◦C.

For amylase activity, a cell-free sample solution (20�g)suitably diluted with 0.02 M Tris–HCl buffer (pH 8.0) was

C. Zhang et al. / Biochemical Engineering Journal 19 (2004) 181–187 183

mixed with 0.4 ml of 0.2% (w/v) potato starch solution (pH10.0 made up with 0.1 M glycine–NaOH buffer) at 40◦C.After 30 min incubation, the reaction was stopped by adding1.0 ml of 0.2 N HCl and 4 ml of 0.005% iodine solution.The absorbance of the sample was measured at 700 nm. Oneunit of the enzyme activity was defined as the amount ofenzyme required to reduce an optical density of 0.32, whichis equivalent to 100�g of potato starch under the aboveconditions.

For assay of cellulase activity, the cell-free sample so-lution (0.5 ml) was mixed with 0.5 ml of 1% (w/v) CMCsolution (pH 6.7 made up of 0.05 M acetate buffer). After30 min incubation at 40◦C, 1 ml of dinitrosalycilic acidsolution was added. The mixture was heated in a boilingwater bath for 5 min, and then 4 ml of water were added.Absorbance of the sample was measured at 510 nm. Oneunit of the enzyme activity was defined as the amount ofenzyme which liberates 1 mg of reducing sugar expressedas glucose per minute under the above conditions.

2.5.3. Assay of enzyme activity in the cellsTo measure the cellulase and amylase activity contained

in microbial cells, the sample was centrifuged to collect thecells. Subsequently an equal volume of de-ionized waterwas added to prepare the solution for assay. Assay of theenzyme activity was carried out by the same method as forcellulase activity. The activity measurements obtained in thisway would include those cells adsorbed on the cell surfaceand/or probably contained inside the cells.

2.5.4. Composition of sweet potatoReducing sugar, starch and cellulose were measured by a

previously reported method[13].

3. Results and discussion

3.1. Mixed alkalophilic culture andmultienzyme production

In order to construct a mixed alkalophilic culture systemto produce multienzymes, the basic culture characteristicsof the constituent strains were examined. For the amy-lase producer B. sp. JCM 9141, amylase activity was onlydetected in the culture broth, and the maximal amylase ac-tivity was 45 U amylase/ml during cultivation with a starchmedium and 50 U amylase/ml during culture with a glucosemedium. The time course profiles of microbial growth andamylase activity in the two media were almost the same(data not shown). Thus, for B. sp. JCM 9141, starch was notan inducer for the production of amylase and both starchand glucose media could produce amylase at the same level.Moreover, amylase production in both of the media was as-sociated with cell growth. But, there were some differencesfound in cellulase production in the CMC medium and glu-cose medium for B. sp. JCM 9156. The cellulase activity in

the CMC medium could be divided into two parts: cellulasein the culture broth (extracellular cellulase) and cellulasecontained in the microbial cells. The maximal total cel-lulase activity was 28 mU cellulase/ml for CMC medium,and 13 mU cellulase/ml for glucose medium, indicating thatglucose as the sole carbon source at high concentrations(>10 g glucose/l) would inhibit the production of cellulasebecause the growth curves in the two media were the same(data not shown). Moreover, extracellular protease activitywas also detected in both of the strains.

During the entire course of mixed cultivation, both ofthe two strains could be observed by microscope, indicatingthat the two constituent strains coexisted stably. Features ofmultienzyme production by the mixed culture are shown inFig. 1. It was obvious that the mixed culture allowed themutienzymes consisting of various amylase, cellulase andprotease to be produced simultaneously in the same flaskculture. Generally speaking,Bacillus species are a popu-lar bacterial family for the production of protease[14]. Inthe present study, alkaline protease was also produced ac-companied with alkaline amylase and cellulase production,which presumably pertained to the spore formation duringthe cultivation. Although until now there has no report onthe production of protease by the two different strains andthe enzymatic property, the protease activities in the presentpaper were measured by the same method, i.e., degradationof casein. As our target was to obtain the mixture of alka-line amylase and cellulase, here, multienzymes consistingof 25 U amylase/ml and 32 mU cellulase/ml could be ob-tained by choosing a time that would achieve the maximalcellulase activity.

Comparisons of the microbial growth and enzyme pro-duction in the mixed culture and the pure cultures are shownin Fig. 2. In all of the experiments, mixed medium obtainedby mixing the starch medium and CMC medium in a vol-ume ratio of 1:1 was used.Fig. 2A shows the growth curvesin the respective pure culture of the cellulase producer, thepure culture of amylase producer and the mixed culture. Theinitial cell densities of the respective alkalophilic strains inthe mixed culture were half of those in the respective purecultures. Thus, in order to examine if the two strains in themixed culture grow with interaction, it was possible to com-pare the measured cell concentration in the mixed culturewith an estimation for the cell concentration in terms ofOD660 in the mixed culture obtained by taking the averagevalue of the cell concentrations of the two strains in theirpure cultures. Obviously, the real total cell concentration inthe mixed culture was higher than the concentration calcu-lated on the basis of the pure cultures. This suggests that inthe mixed culture, the two strains grew interactively. To un-derstand the individual bacterial growth and the interactionin the mixed culture, monitoring the respective cell densitiesof the two strains is needed. As the traditional method is diffi-cult to measure the respective bacterial concentrations in themixed culture, new technique for tracing the different strainsis needed. We are now developing a rapid quantification tool

184 C. Zhang et al. / Biochemical Engineering Journal 19 (2004) 181–187

0 20 40 60 80 100

-2

0

2

4

6

8

10

12

14

16

18

20

22

24

26

28

30

32

34

time (h)

cellu

lase a

ctivity(m

U c

ellu

lase/m

l)

0

10

20

30

40

50

60

70

pro

tease a

ctivity(U

pro

tease/m

l)

am

yla

se a

ctivity(U

am

yla

se/m

l)

0

10

20

30

40

50

60

70

80

90

100

110

120

130

Fig. 1. Time course profiles of extracellular enzymatic activity during the mixed culture with the mixed media. Cellulase (�); amylase (�); protease (�).

0 20 40 60 80 100

-10

0

10

20

30

40

50

60

70

80

am

yla

se

activity(U

am

yla

se

/ml)

time(h)

time(h)

0 20 40 60 80 100

1

2

3

4

5

6

7

OD

66

0(-

)

time(h)

0 20 40 60 80 100

-5

0

5

10

15

20

25

30

35

40

45

50

ce

llula

se

activity(m

U c

ellu

lase

/ml)

time(h)

20 30 40 50 60 70 80

0

50

100

150

200

250

pro

tease a

ctivity(U

pro

tease/m

l)

(B) (A)

(C) (D)

Fig. 2. Comparisons of the microbial growth and enzyme production in the mixed culture and the pure cultures. (A) Growth curves of mixed cultureof amylase producer and cellulase producer (�), pure culture of amylase producer (�), pure culture of cellulase producer (�), average value of cellconcentration when the two strains would grow together without interaction (�). (B) Extracellular amylase activity of the mixed culture (�) and pureculture of amylase producer (�). (C) Cellulase activity (total= extracellular) of the mixed culture (�), total cellulase activity (�) and extracellularcellulase activity (�) of pure culture of cellulase producer. (D) Extracellular protease activity of the mixed culture (�), pure culture of amylase producer(�), pure culture of cellulase producer (�), average value of cell concentration value when the two strains would grow together without interaction (�).

C. Zhang et al. / Biochemical Engineering Journal 19 (2004) 181–187 185

for analyzing the mixed culture by labeling each constituentstrain with fluorescence proteins of different colors.

To examine the characteristics of multienzyme productionby the mixed culture, a comparison of amylase, cellulaseand protease production in the pure cultures and the mixedculture was made. The amylase activity produced by themixed culture was a little higher than the pure culture ofthe amylase producer (Fig. 2B). Fig. 2Cshows the cellulaseactivity. Although the total cellulase activity (extracellularactivity and activity contained in the microbial cells) inthe mixed culture was slightly lower than that of the pureculture, the extracellular enzyme activity was much higherin the mixed culture than in the pure culture. This resultindicated that the mixed alkalophilic culture enabled thesecretion of alkaline cellulase from the microbial cells intothe culture broth to be enhanced.Fig. 2D shows the com-parison of protease activity measurements in the respectivepure cultures and in the mixed culture. The protease activitywas also calculated by the same method as mentioned abovefor estimations of the two strains’ growth. The calculatedaverage value of protease activity in the two pure cultureswas higher than that produced in the mixed culture, whichmeans that protease production was reduced in the mixedalkalophilic culture compared with the pure cultures. Thismight be ideal for the production of the targeted multien-zymes because the protease would be harmful to the othertwo enzymes. Incidentally, the errors for the measurementof cell concentration and the respective enzymatic activitieswere less than 3%, and the same cultures done at differentruns showed almost same trends.

Even in the medium containing starch or CMC as the solecarbon source, only the final product of glucose formed fromthe polymer hydrolysis by bacterial amylase or cellulase canbe utilized for cell growth. Glucose is favorable for bothgrowth and amylase production by the amylase producer,while it inhibited the cellulase production by the cellulaseproducer at high concentrations, as mentioned above.Fig. 3shows changes in glucose concentration in the pure culturesand mixed culture with glucose and mixed media. Resultsfrom the pure cultures of the two strains with the glucosemedia indicate that glucose consumption rate by the amy-lase producer was much higher than that by the cellulaseproducer. In comparison, the glucose concentration in themixed culture with the mixed medium remained at around

Table 1Recovery of different crude enzymes

Items Enzymes Activities before EtOHprecipitation (U/ml)

Activities after EtOHprecipitation (U/ml)

Recovery times Recovery ratio(% U/U)

Pure enzymes Amylase 77.8 176.9 2.5 91.0Cellulase 21.8× 10−3 31.4 × 10−3 2.5 57.6

Multienzymes Amylase 14.1 10.5 2.5 29.8Cellulase 24.7× 10−3 30.4 × 10−3 2.5 49.2

Mixed enzymes Amylase 58.6 36.9 2.5 25.2Cellulase 17.3× 10−3 5.7 × 10−3 2.5 13.2

0 20 40 60 80 100 120

-1

0

1

2

3

4

5

6

7

glu

co

se

co

nce

ntr

atio

n (

mg

glu

co

se

/ml)

time (h)

Fig. 3. Time course profiles of glucose concentration under various cultureconditions. Pure culture of amylase producer in glucose medium (�);pure culture of cellulase producer in glucose medium (�); pure cultureof cellulase producer in mixed medium (�); mixed culture in mixedmedium (�).

1 g glucose/l after 24 h cultivation, which was just a littlelower than that in the pure culture of the slow-growing cel-lulase producer with the same medium. This suggested thatglucose hydrolyzed from CMC and starch by cellulase andamylase in the mixed culture was sufficient for the growthof both the amylase and cellulase producers. These resultssuggested that the microbial interaction between the amy-lase and cellulase producers and/or their competition to theglucose consumption in the mixed culture could pertain tothe differences in the enzyme production and cell growthas shown inFig. 2. Detailed experiments to explain theseresults are underway.

3.2. Recovery of multienzymes by ethanol precipitationand its application

Multienzymes were recovered by the conventional ethanolprecipitation method. The recovery ratios of different en-zymes are listed inTable 1. Here, the mixed enzymes wereprepared by mixing the pure enzymes of alkaline amylaseand cellulase obtained from the respective pure cultures atan activity ratio of 1:1 directly. The respective initial enzyme

186 C. Zhang et al. / Biochemical Engineering Journal 19 (2004) 181–187

-1 0 1 2 3 4 5 6 87

0.0

0.5

1.0

1.5

2.0

2.5

Incre

acm

en

t in

glu

co

se

co

nce

ntr

atio

n(m

g g

luco

se

/ml)

time(h)

Fig. 4. Increments in glucose concentration in enzymatic treatment of sweet potato with additions of filter paper and CMC. Only filter paper treated bymultienzyme (0.8215 U amylase and 0.2 U cellulase/g sweet potato) (�); CMC + sweet potato treated by multienzyme (0.8215 U amylase and 0.2 Ucellulase/g sweet potato) (�); CMC + sweet potato treated by single amylase (0.8215 U amylase/g sweet potato) (�).

activity was kept at the same level in all of the experiments.The recovery ratios of the multienzymes and the mixed en-zymes were much lower than the respective pure ones. Thismight be due to the interaction of the two enzymes duringthe ethanol precipitation, which needs to be studied further.

The effect of the recovered multienzymes on the saccha-rification of starch was examined by using sweet potatoes.The sweet potato was chosen because it contains both starchand cellulose and is an important crop in China. The compo-sition of the sweet potato used in the study included (on dryweight basis): 57.46% (w/w) starch, 3.80% (w/w) celluloseand 1.94% (w/w) reducing sugar.

The saccharification of the sweet potato was performed atpH 10 as described inSection 2. Incidentally, the enzymaticsaccharifying ratio of the sweet potato can reach 28% in 6 hwithout any optimization.

Multienzymes and amylase showed almost the same trendfor the saccharification of sweet potato (data not shown),which indicated that the recovered multienzymes showedthe same activity levels as pure amylase, while the cellulaseexerted no action on the cellulose contained in the sweetpotato. Moreover, amylase and mixed enzymes (mixingamylase and cellulose together) showed the same result onthe sacchrification of the sweet potato (data not shown). Toexamine this in detail, an experiment with the enzyme treat-ment for filter paper and water-soluble CMC was also carriedout and the results are shown inFig. 4. Almost no degrada-tion of the filter paper by the multienzymes was observed,while in the experiment for the mixture of the sweet potatoand CMC, multienzymes showed higher glucose concentra-tion than for the single amylase. These results suggested that

cellulase produced in our study could act as soluble celluloserather than as insoluble cellulose. In practical use of the mul-tienzymes to starch processing of sweet potatoes, cellulase’scapability of acting on insoluble cellulose is important, andthis property of cellulase is currently being studied.

Alkaline amylase and cellulase are expected to be usefulin the clean processing of starch from crops, because thephysical solubilization of starch at the alkaline conditionand the enzymatic actions can occur simultaneously. Be-cause extracellular enzymes are advantageous in bioprocessengineering, the mixed alkalophilic culture is a promisingcandidate for use in the production of extracellular alkalinecellulase and amylase. As shown in this study, through themixed culture of the two producers, two enzymes were ob-tainable in only one culture with higher activity levels. Inaddition, purifying the two enzymes at one time will alsobe environmentally friendly compared to the conventionalprocess for the production of a single enzyme.

Acknowledgements

This work is supported by the Grant of National NaturalScience Foundation of China (No. 20176025).

References

[1] J.E. Bailey, D.F. Ollis, Biochemical Engineering Fundamentals,McGraw-Hill, New York, 1986.

[2] K. Miyano, K. Ye, K. Shimizu, Improvement of Vitamin B12fermentation by reducing the inhibitory metabolites by cell recyclesystem and a mixed culture, Biochem. Eng. J. 16 (2000) 207–214.

C. Zhang et al. / Biochemical Engineering Journal 19 (2004) 181–187 187

[3] M. Tohyama, Y. Patarinska, Z. Qiang, K. Shimizu, Modeling of themixed culture and periodic control for PHB production, Biochem.Eng. J. 10 (2002) 157–173.

[4] T. Mitsue, K. Tachibana, Y. Fujio, Efficient Kefiran productionby a mixed culture ofL. kefiranofaciens KF-75 and yeast strains,Seibutsu-Kogaku Shi. 77 (1999) 199–203 (in Japanese).

[5] J.P. Delgenes, M.C. Escare, J.M. Laplace, R. Moletta, J.M. Navarro,Biological production of industrial chemicals, i.e. xylitol and ethanol,from lignocelluloses by controlled mixed culture systems, Ind.Crop. Prod. 7 (1998) 101–111.

[6] T. Fujio, A. Maruyama, Y. Aoyama, S. Kawahara, T. Nishi,Production of 5′-GMP from glucose by coupling reaction betweenCorynebacterium ammoniagenes and self-clonedEscherichia coli,Seibutsu-kogaku Shi. 77 (1999) 104–112 (in Japanese).

[7] T. Fujio, A. Maruyama, H. Mori, New production methods for usefulsubstance using ATP regeneration system, Bios. Ind. 56 (1998) 7373–7442 (in Japanese).

[8] H.S. Olsenl, P. Falholt, The role of enzymes in modern detergent,Detergent Cosmetics 23 (2000) 6–9 (in Chinese).

[9] A.C. Furlan, M. Fraiha, A.E. Murakami, E.N. Martins, C. Scapinello,I. Moreira, Utilization of a multienzyme complex in diets containingtriticale for broilers. 1. Digestibility assay, Revista de SociedadeBrasileira de Zootechia-J. Braz. Soc. Anim. Sci. 26 (1997) 759–764.

[10] Y.L. Dong, K.W. Wen, B.C. Li, X. Gao, G.Y. Sheng, J.M. Fu,Investigation on nitrogen nutritive value of cheap sea fishEngraulisjaponicus, Food Ferment. Ind. 26 (2000) 28–30 (in Chinese).

[11] K. Horikoshi, Production of alkaline enzymes by alkalophilicmicroorganisms. Part II. Alkaline amylase produced byBacillus no.A-40-2, Agric. Biol. Chem. 35 (1971) 1783–1791.

[12] K. Horikoshi, M. Nakao, Y. Kurono, N. Sashihara, Cellulases of analkalophilic Bacillus strain isolated from soil, Can. J. Microbiol. 30(1984) 774–779.

[13] Y.X. Zhai, Handbook of Food Analysis, Peking University, Beijing,1988 (in Chinese).

[14] K. Horikoshi, Production of alkaline enzymes by alkalophilicmicroorganisms. Part I. Alkaline protease produced byBacillus no.221, Agric. Biol. Chem. 35 (1971) 1407–1414.