TỐI ƯU HOÁ QUÁ TRÌNH THUỶ PHÂN BÃ NẤM MEN BIA BẰNG PHƯƠNG PHÁP TUẦN HOÀN LIÊN TỤC

Response surface methodology (RSM) [9] was used to determine optimum condition for continuous circulation proteolytic hydrolysis of spent brewer’s yeast by using Alcalase.[r]

OPTIMIZATION FOR PROTEOLYTIC HYDROLYSIS SPENT BREWER’S YEAST BY CONTINUOUS CIRCULATION METHOD

Nguyen Thi Thanh Ngoc * , Dinh Van Thanh, Dinh Van Thuan

East Asia University of Technology

ABSTRACT

A large amount of spent yeast is generated from brewing industry as a by-product with high- value source of protein (about 50-55% protein) and the hydrolysate from spent brewer’s yeast have been found many applications in food technology The yield of proteolylic hydrolysis for spent brewer’s yeast and amino acid contents of hydrolysates depend on technological factors such as temperature, pH value, type of used enzyme and ratio enzyme/substrate, hydrolysis time and hydrolysing methods (batch-, or continuous method) In this study, with the purpose to hydrolyze the spent brewer’s yeast for food application in industrial scale, it was used continuous circulation method Response surface methodology (RSM) was used to determine optimum condition for continuous circulation proteolytic hydrolysis of spent brewer’s yeast The optimal conditions for obtaining high degree of hydrolysis were: Ratio of enzyme mixture (alcalase): 9.0 U/g, pH: 7.5, percentage of intverter’s pump: 65%, hydrolysis temperature: 55 o

C and time: 9 hours and the yield

of hydrolysis reached value 56.83% ± 0.51

Keywords: optimization, continuous circulation, proteolytic hydrolysis, degree of hydrolysis,

brewer’s yeast

INTRODUTION **

Spent brewer’s yeast, the by product from the

brewing industry, is being produced in large

amount annually from beer manufacturers due

to the increasing volume production [1] It is

generally used primarily as inexpensive

animal feed after inactivation by heat and

much of this by product is considered

industrial organic waste that causes a great

deal of concerns Such wastes are generally

incinerated or put into landfill, in which case,

remaining proteins and amino acids, and other

useful substances were not recovered [2] In

addition, incineration of organic waste often

gives toxic emission whose distribution

degree is even higher than that of organic

solid waste Attempts have been made to

recover higher value protein and amino acid

products from spent Brewer’s yeast [3] by

employing various processes such as

autolysis, plasmolysis [4], acid or alkali

catalyzed hydrolysis, or enzymatic hydrolysis

[5, 6], overflow or continuous methods

Review of published researches to date

indicates there are several problems in this

* Tel: 0989 965295, Email:ngoc.nguyen@eaut.edu.vn

area One is the high cost of using large quantities of enzyme in batch – type operations [7] and long time hydrolysis (the

microorganisms) The second is energy and labor cost in production the last, equipment may require considerable floor space Leading

to resulting in low yields and/or poor productivity [7, 8] So that, the scientific aims

of this study is to describe the design and performance of continuous hydrolysis of yeast’s protein by a continuous circulation method Response surface methodology (RSM) [9] was used to determine optimum condition for continuous circulation proteolytic hydrolysis of spent brewer’s yeast

by using Alcalase

MATERIALS AND METHODS

Materials

The spent brewer’s yeast Saccharomyces used

as a substrate was donated by brewer’s Sai Gon Ha Noi Flavourzyme and alcalase were obtained from Novozymes, Denmark Proteolytic activity is 289 U/g and 328 U/g Flavourzyme is a food grade exoprotease

from Aspergillus oryzae, its main enzyme

component is EC 3.4.11.1 Alcalase is a food

grade endoprotease from Bacillus

licheniformis, its main enzyme component is

the serine protease subtilsin A (EC 3.4.21.62)

Methods

Washing process: Spent brewery’s yeast was

washed once with NaOH 0.1N for removing

polyphenols and 2 times with cold water for

the removing remained solids, and then

centrifuged at 4000 rpm at 4 oC for 15 min

using a thermo Fisher (USA) to recover

solids, which were material for further

studies

Pretreament yeast cell: Sludge of treated

yeast was heated shock process (The first

time of the heat shock process is from 1 to 3

minutes at 68oC, then incubated for 1 hour at

45-50oC and the second time to heat the

process from 1 to 3 minutes at 68oC, then

incubate for 1 hour at 52 - 55oC [6] After

heated shock process sludge’s yeast was

adjusted to pH 5.5 (using HI 2211 pH/ORP

meter) by NaOH 0.2N The ratio of yeast:

water was 1:1.5 (w/w), and autolysis was

carried out at 50oC in 24 hours

Figure 1 Diagram of continuous circulation

hydrolysis (1 Sludge yeast tank; 2 Hot water tank;

3 Tube heat exchanger (include 30m tube DN 25

and 36m tube DN 32); 4 pH; 5 Motor for paddle; 6

and 15 Thermal sensor; 7 and 14 Heating bar; 8

Loadcell; 9 Circulation pump; 10 Flowmetter; 11

Enzyme pump; 12 NaOH pump

Hydrolysis process: After autolysis process,

autolysate was adjusted and added enzymes

(Alcalase) and then continuous hydrolysis

process was performed on continuous

circulation system (Fig 1) using agitator with

agitation speed (M) 250 rpm under different

conditions Autolysate was continuous circulation between tank (1) and tube heat exchanger (2) by pump (9) The sample was inactivated by 0.5 M TCA and the sludge was removed by using centrifuge (6000 rpm, at

4oC for 10 min)

Determination of degree of hydrolysis: In

protein hydrolysis, the key parameter for monitoring the reaction is the degree of hydrolysis (DH), which is determined as the percentage of amino acids before and after hydrolysis process for spent brewery’s yeast The following formula was used for calculation [10]: DH = Ns/Nt× 100%; - where

Ns is amino acid content in hydrolysate, it was determined by Ninhydrin method using glutamic standard (Merck KgaA, Germany)

Nt is the total nitrogen content in yeast dry before hydrolysis, it was measured by the Kjeldahl method

optimization: Experimental design: The

response surface method with CCOD (central composite orthogonal design) were used to study the effects of independent factors: E/S ratio of Alcalase, temperature, pH, time of hydrolysis and % inverter’s pump (9) Desirable responses are the followings: Degree of hydrolysis (Y1, %) (Table 1) This design has 50 trials including 32 trials for factorial design, 8 trials for axial points and

10 trials for central points (Table 2) Optimization: For predicting the optimal point, second-order polynomial models were fitted to correlate relationship between independent variables and response CCOD was performed to evaluate the optimal operating conditions to obtain maximum DH

of hydrolysis

Statistical analysis: Design Expert software

version 10.0 (Stat-Ease, Minneapolis) was used for the regression analysis of experimental data, to plot response surface and to optimize by desirability methodology

RESULTS AND DISCUSSION

Model building and statistical significance test

Table 2 shows the process variables and

experimental data of 50 runs The

experimental results were fitted with a

second-order polynomial equation by a

multiple regression analysis

Analysis of variance for models is shown in

Table 3 F-value models is 2552.05 (Y1) It is

indicated that all the regression model is

highly significant at confidence level of

99.99% (p<0.0001) The indicates coefficient

is significant if the p-value is less than 0.05

As it is shown in this table, confidence level

with p < 0.0001 (excepting a cross coeffecient

of AC, AD, BC and CD in Y1 F-value for

lack of fit of Y1 model is 1.179 (p = 0.4394)

The models were fit with experiment

Moreover, the coeffecients of determination

(R2) of the models is 0.9999 (Y1), indicating

that 99.99% of variability in the response

could be predicted by the models The models

for the response variables could be expressed

by the following second – degree model in

terms of coded factors

Y1 = 68,61 + 1,11A – 0,79B + 0,6C + 0,66D

+ 2,26E – 0,31AB + 0,03AC – 0,2AD –

0,23AE - 0,04BC - 0,54BD – 0,61BE -

0,07CD – 0,25CE – 0,31DE – 8,79A2 –

7,49B2 - 2,86C2 - 2,45D2 – 4,7E2

Considering in turn the effect of each factor

(when others are fixed at zero level) on the

DH (Fig.2), it shows that hydrolysis

temperature (A) and pH (B) significantly

affect the overall DH (Y1); whereas, E/S ratio

and hydrolysis time are the less significant

factors this result is the similar to the study

by Tavano [6] The effects of temperature and

pH on DH is possiblely due to their impact

on the catalytic activity of the enzyme The effects of temperature and pH on the response surface of Y1 function were showed more detail in Fig.3

Optimization and verification of the models

The algorism of fastened targets according to desirability methodology invented by Derringer and et al [8] was applied The optimum parameters of DH of protein hydrolysis from spent brewer’s yeast as follows: E/S ratio (Alcalase 9.0U/g), pH 7.5, hydrolysis temperature 55oC, hydrolysis time 9.0 hours, level of inverter’s pump 65% Under the optimal conditions, the corresponding response value predicted for the final DH 57.29% The final DH has achieved of 99.34%, 100% and 99.90% desirability of proposed objective, respectively (Fig.4)

In order to confirm the predicted results, the hydrolysis conditions (ratio E/S: 9.0U/g, pH: 7.5, temperature: 55oC, time: 9 hours) were sellected in the experiments (five times) The mean value of the maximum DH have reached 56.83% ± 0.51, (Table 4) There was

a good coordination between the observed and the predicted values in models The result

of DH in this study is higher than those by Chae H J, Joo H, 2001[1] (DH obtained 48.3% when the yeast cells were treated using

a mixture of 0.6% Protamex and 0.6% Flavourzyme

Table 1 The variables and their levels of the Hydrolysis

Variables Symbols units Symbolic coding value

Level of

Table 2 Experimental design and results

Exp

No

A

( o C) B

C (U/g)

D (hour)

E (%)

Y 1

(%)

Exp

No

A ( o C) B

C (U/g)

D (hour)

E (%)

Y 1

(%)

Table 3 Regression analysis of overall DH Y 1

Mean Square F value p-value (Prob > F) Model 288.758 2552.05 < 0.0001

Lack of Fit 0.11746 1.17917 0.4394

Figure 2 The Influence of factors on DH Figure 3 Response surface plot of protein

hydrolysis process for DH

Figure 4 Responsible desirability level Table 4 The verifying results the compatibility of the model with experimental

Number Temperature

(oC)

pH E/S ratio

(U/g)

Time (hour)

Level of inverter’s pump (%)

DH (%)

According to

experiments

0.51 CONCLUSIONS

The statistical experimental design using the response suface and desirability methodology to optimize the process paremeters of the continuous circulation proteolytic hydrolysis of spent brewer’s yeast by using proteases The optimum conditions: the E/S ratio: 9.0 U/g (Alcalase) pH: 7.5 temperature: 55oC time: 9 hours and the level of inveter’s pump 65% The similarity of the value of DH between the experiment and the predicted using the models under these conditions indicated that the models are satisfactory and accurate

Acknowledgement

We thank to School East Asia University of Technology and my office’s staff

REFERENCE

1 Chae H J Joo H (2001) Utilization of

brewer's yeast cells for the production of

food-grade yeast extract Bioresour Technol 76 pp

253-258

2 Zhang Ji Wang Junjie Chen Keyu Chu Can

(2008) Study on production of yeast extract from

beer yeast China Brewing 15: 26-29

3 Adler N J (1976) Enzymatic hydrolysis of

proteins for increased solubility J Agric Food

Chem 24 1090 -1093

4 Tatiana Vukasinovic Milic Marica Rakin and

Slavica Siler - Marikncovic (2006) Utilization of

baker’s yeast for the production of yeast extract:

Effects of different enzymatic treatments in solid

protein and carbohydrate recovery Faculty of

Technology and Metallungry Karnegijeva 4

Belgrade Serbia 4: 296-378

5 Bayarjargal M Munkhbat E Ariunsaikhan

T Odonchimed M Uurzaikh T Gan E.T

Regdel D (2011) Utilization of spent brewer’s

yeast Saccharomyces cerevisiae for the production

of yeast enzymatic hydrolysate Mongolian J Chem 12 88 -91

6 Tavano OL (2013) Protein hydrolysis using proteases an important tool for food

biotechnology J Mol Catal 90: 1-11

7 Cheftel C Ahren M Wang D.I.C Tannenbaum S.R (1971) Enzymatic solubilization of FPC: Batch studies applicable to

continuous enzyme recycling process J Agric Food Chem 19 155

8 Derringer G Suich R (1980) Simultameous

optimization of serveral responses variables J Qual Techol 12 214-219

9 Dougherty D A (2006) Unnatural amino acids as probes of protein structure and function

Chem Biol 4 645-652

10 Haefeli R.J Glaser D (1990) Taste responses and thresholds obtained with the

primary amino acids in humans Lebensm-Wiss U- Technol 23 523-527

TÓM TẮT

TỐI ƯU HOÁ QUÁ TRÌNH THUỶ PHÂN BÃ NẤM MEN BIA BẰNG PHƯƠNG PHÁP TUẦN HOÀN LIÊN TỤC

Nguyễn Thị Thanh Ngọc *

Đinh Văn Thành Đinh Văn Thuận

Trường Đại học Công nghệ Đông Á

Lượng lớn bã nấm men bia từ các nhà máy bia công nghiệp là nguồn protein có giá trị cao (khoảng

50 – 55%) và dịch thuỷ phân bã nấm men bia có nhiều ứng dụng trong công nghệ thực phẩm Hiệu suất quá trình thuỷ phân cũng như thành phần acid amin trong dịch thuỷ phân phụ thuộc vào các yếu tố công nghệ như nhiệt độ pH loại enzyme và tỷ lệ enzyme/cơ chất thời gian thuỷ phân và kỹ thuật thuỷ phân (kỹ thuật theo mẻ hoặc liên tục) Trong nghiên cứu này với mục đích ứng dụng sản phẩm thuỷ phân trong công nghệ thực phẩm ở quy mô công nghiệp nên hệ thống thuỷ phân tuần hoàn liên tục được sử dụng Phương pháp bề mặt đáp ứng được sử dụng để xác định điều kiện tối ưu quá trình thuỷ phân tuần hoàn liên tục bã nấm men bia Điều kiện tối ưu cho mức độ thuỷ phân cao nhất là: tỷ lệ enzyme (alcalase): 9.0 U / g pH: 7.5 nhiệt độ: 55oC thời gian: 9 giờ mức cài đặt biến tần của bơm: 65% và mức độ thuỷ phân đạt được là 56.83% ± 0.51

Từ khóa: tối ưu hoá tuần hoàn liên tục thuỷ phan protein mức độ thuỷ phan bã nấm men

Ngày nhận bài: 28/8/2018; Ngày phản biện: 30/8/2018; Ngày duyệt đăng: 31/8/2018

*