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Bioresource Technology 60 (1997) 101-106 © 1997 Elsevier Science Limited

All rights reserved. Printed in Great Britain 0960-8524/97 $17.00

RECYCLING OF BAKERY WASTES USING AN AMYLOLYTIC LACTIC ACID BACTERIUM

Yuji Oda , a* Bang-Sun Park/' Kook-Hyun Moon c & Kenzo Tonomura"

"Department of Food Science and Technology, Fukuyama University, Fukuyama, Hiroshima, Japan h College of Home Economics, Chung Ang University, San 40-1, NeoRi, Dedug-Myun Ansung-Kun, Kyungki-Do, Korea

"Yuhan-Kimberly Ltd, 157-33 Samsung Dang, Kangnam-Ku, SeouL Korea

(Received for publication 24 January 1997)

Abstract Production of lactic acid from discarded bread by using an amylolytic lactic acid bacterium, Lactoba- cillus amylovorus, was investigated to recycle bakery wastes. Addition of 2.0% yeast extract in the medium containing 3.58% bread crust caused maximum acid production. The stimulation of lactic acid production by less expensive materials such as corn steep liquor, defatted soybean powder, rice bran and wheat bran at additional levels of 2.0% was also observed, but limited. Acid production in the medium supplemented with 2.0% corn steep liquor was enhanced by the fur- ther addition of 2.0% defatted soybean powder and reached the levels comparable to medium containing 1.4% yeast extract. In the medium supplemented with 2.0% corn steep liquor and 2.0% defatted soybean powder, 47.2% of total sugars was converted to OL- lactic acid in 72 h under static incubation. When the baking test was carried out, the bread made with the addition of the culture filtrate was significantly (P < 0.05) preferred over those with and without addi- tion of commercial fermented seasoning with respect to taste and overall acceptability. © 1997 Elsevier Science Ltd.

Key words: Bread crust, lactic acid fermentation, Lactobacillus amylovorus, seasoning for breadmak- ing, sensory test.

INTRODUCTION

Various types of baked foods are made and con- sumed in many countries of the world (Pomeranz, 1987). About 1.2 tons of wheat flour are consumed in Japan yearly for the production of bread and rolls, and white bread dominates the total bread market. During the manufacturing process of the bread, substandard bread and the bread crust removed to make special types of sandwich breads are constant by-products. Furthermore, all of the

*Author to whom correspondence should be addressed. 101

baked products not sold in the retail stores are returned to the manufacturer. These by-products and unconsumed breads, as described above, consti- tute a major portion of the wastes from the baking industry. The amount of waste is not reported, but it may reach at least 1% of the total amounts of flour consumed in this industry. Although unconsumed bread contains variable amounts of sugar, fat and milk derived from different baked products, the fundamental constituent is starch. Due to this abun- dance and renewable resource, we have investigated the conversion of the starch in bakery wastes to lactic acid without supplementing starch-degrading enzymes.

Lactic acid is widely used in the food industries (Duxbury, 1992; Duxbury, 1993). This organic acid exhibits preservative properties; inhibiting microbial spoilage in meats, sea foods, mayonnaise, salad dressing and soft drinks. Emulsifiers derived from lactic acid improve the quality of breads, cake mixes, fillings and toppings, powdered coffee, shortenings and whiteners. This acid functions as a flavor enhan- cer in beer, wine, cider, soft drinks, candies, frozen desserts, jams and jellies, margarine and pickles. Recently, polylactide polymers made from lactic acid are anticipated to become potential biodegradable plastics (Ozeki and Fujii, 1994). Expanded use of lactic acid has elicited more attention regarding its biotechnological production (Lipinsky, 1981).

The present paper reports the optimization of the production of lactic acid from bread crust and the application of the culture filtrate obtained from the lactic acid fermentation in the breadmaking process as an economical method of recycling bakery wastes.

METHODS

Materials Bread crust containing starch at 72.4% of a dry weight was obtained from a local bakery. Yeast extract (YX, Nakarai-Tesque, Inc., Kyoto, Japan), corn steep liquor (CSL Nippon Denpun Kogyo Co.,

102 Y Oda, B.-S. Park, K.-H. Moon, K. Tonomura

Kagoshima, Japan), defatted soybean powder (DSP), rice bran (RB) and wheat bran (WB) are commer- cially available products. Seasoning based on fermented milk (FOUR-MATE TM) was a gift from Kyowa Hakko Kogyo Co. Ltd (Tokyo, Japan).

Organism and culture Lactobacillus amylovorus JCM 1127 [NRRL B-4532] (Nakamura, 1981), an amylolytic lactic acid bacte- rium, was obtained from the Institute of Physical and Chemical Research (Wako-shi, Saitama, Japan). Unless otherwise stated, the bacterial cells were grown for 24 h in 5 ml of MRS medium (De Man et al., 1960) in which glucose had been substituted by starch. One millilitre of this culture was inoculated to 100 ml of basal medium containing 3.58% bread crust on a dry basis, supplemented with YX, CSL, DSP, RB or WB and incubated for 72 h at pH 6.0. All of the cultures were subjected to cultivation tem- peratures of 37°C under static incubation.

The culture broth was centrifuged at 20,000 x g for 10 rain to separate the supernatant from the pre- cipitate. The precipitate was washed twice with distilled water when necessary. Alternatively, the culture broth was passed through three layers of cheese cloth to obtain culture filtrate containing viable cells of the lactic acid bacterium.

or the commercial fermented seasoning at levels of 15 g each. After fermentation for 1 h at 30°C, the dough was cut into 10 pieces of 35 g each, proofed at 37°C and 85% relative humidity. When the dough had risen to a height of 3.0-4.7 cm, the dough was subjected to the molding and panning stages, and then baked at 170°C.

After storage at room temperature for 1 day in a polyethylene bag, each one of the four pieces cut from the baked bread was coded and served, in a random order, to a panel consisting of 12 student members from the Department of Food Science and Technology at Fukuyama University. Care was taken to use the same panelists for each test. Flavor, taste and overall acceptability were evaluated by the rank- ing test, and the sums of rank positions (1 = first, 2 = second, 3 = third) were statistically analyzed (Jellinek, 1985).

Reproducibility All the experiments were independently carried out three times. The data are reported as the average values and standard deviations obtained from these experiments.

RESULTS

Analytical methods The volume (ml) of 0.1N NaOH required to neutralize free acids in 1.0 ml of the supernatant was recorded as acidity. Lactic acid was determined by the enzymatic determination kits (Boehringer Man- nheim, Germany). Reducing sugars were measured by the 3,5-dinitrosalycylic acid method (Bernferld, 1955) after hydrolysis of starch in 0.7 N HCI at 100°C for 2.5 h.

Baking test and sensory evaluation The dough for white bread was made by mixing 300 g wheat flour, 6 g NaCI, 15 g sucrose, 9 g short- ening, 0.3 g yeast food, 186 ml water and 9 g of a commercial compressed yeast (moisture 67%) with and without the addition of either the culture filtrate

Since bread crust is mainly composed of starch, the lactic acid bacterium might additionally require some nitrogen compounds for its growth in the present experiments. We have focused on evaluating several agricultural by-products for this purpose.

Lactobacillus amylovorus JCM 1127 was grown in the basal media supplemented with 2.0% of either YX, CSL, DSP, RB or WB, and an increase in acidity of the supernatant during cultivation over a 72 h period was measured (Fig. 1). YX elevated the acid production most efficiently among the com- pounds tested. When the differences of acidities in the presence or absence of the other nitrogen com- pounds were compared, CSL, DSP, RB and WB, in order, appeared to stimulate acid production. Some strains included in Lactobacillus other than L. amy-

1.5

,~ 1

0.5

None YX CSL DSP RB WB Addition

Fig. 1. Effect of YX, CSL, DSP, RB and WB each at 2.0% on acid production in the basal medium. Open and closed bars mean acidities in the presence and absence of 3.58% bread crust, respectively.

Lactic acid fermentation 103

lovorus are reported to degrade starch (Fitzsimons and OConnell, 1994). We have preliminarily screened such bacteria and found several strains in the vegetables pickled in rice-bran pastes. Although the isolated bacteria were not identified, the acid production by these microorganisms from bread crust was less than one third of that by the strain JCM 1127 (data not shown).

Figure 2 shows acid production in the media sup- plemented with various amounts of YX and CSL. Each compound enhanced the acidity with an increase in concentration, but CSL never surpassed YX in effect on acid production. Although YX has been shown to be the superior compound, some problems arise when it is used for industrial produc- tion of lactic acid from bread crust. First, YX is an expensive material priced at ¥800-¥2,800/kg which is more than 10 times the cost of the other four compounds. Secondly, the distinct flavor and taste of YX may diminish the quality of baked bread if the culture filtrate supplemented with YX is added to the dough formula.

Then, either CSL, DSP, RB or WB at 2.0% was further added to the medium supplemented with 2.0% CSL (Fig. 3). The further addition of DSP, RB or WB almost doubled the acidity level, while CSL

had little effect. Since RB and WB appreciably ele- vated the acidity in the absence of bread crust, CSL and DSP may be the best combination. The effect of 2.0% CSL plus 2.0% DSP corresponded to that of 1.4% YX. When the bacterium was cultured con- taining various amounts of bread crust, supple- mented with 2.0% CSL and 2.0% DSP, the acidity increased with the increase in concentration and reached a maximum of higher than 3.58% (Fig. 4).

Since the culture medium containing insoluble bread crust is a heterogeneous system, the growing cells are not in constant contact with the starch due to the nature of the static cultivation conditions. However, the acidity increased gradually during cul- tivation and reached a constant value after 120 h (Fig. 5), indicating that lactic acid fermentation pro- ceeded to a certain level under these conditions. The cells of L. amylovorus JCM 1127 may efficiently utilize the sugars liberated after the hydrolysis of starch by the extracellular amylases released into the medium.

Culture broth supplemented with 2.0% CSL and 2.0% DSP was fractionated as described in the Methods. The precipitate was washed and the washed solution was incorporated into the super- natant. Table 1 shows the amounts of total sugars

f J

J

r" /

J

i t - - - - -4

0 , I , I i I , I

0 1 2 3 4 Concentration (%)

Fig. 2. Effect of YX (- - -) and CSL (--) on acid production in the basal medium containing 3.58% bread crust.

1.5

0 .5

--t-

C S L D S P R B W B

A d d i t i o n

Fig. 3. Effect of CSL, DSP, RB and WB each at 2.0% on acid production in the basal medium supplemented with 2.0% CSL. Open and closed bars mean acidities in the presence and absence of 3.58% bread crust, respectively.

104 Y Oda, B.-S. Park, K.-H. Moon, K. Tonomura

1.5

0.5

0 I

0 2

Fig. 4. Effect of bread crust on acid production in the basal

i I i I

4 6 8

Concentration (%) medium supplemented with 2.0% CSL and 2.0% DSP.

and D- and L-lactic acid in both fractions. Soluble sugars and lactic acid present in the supernatant before cultivation are probably derived from the bread crust and CSL, respectively. Although the dif- ference in total sugars was 42.7% of the initial amount, the lactic acid concentration of the super- natant (134 rag/l) was higher than the amount found in commercial seasonings (83 mg/l) sold to the bak- ing industry. The amount of total sugars in the supernatant did not change dramatically during cul- tivation. This phenomenon may be explained by the

presence of intermediate products derived from starch hydrolysis.

The culture filtrate obtained was pale brown with a slight sour taste and included viable cells of the lactic acid bacterium at about 7 x 107 colony-forming units/ml. This filtrate was mixed with the dough ingredients to make white bread and its effect on the bread was compared with that of the commercial seasoning. Few differences were found in the pro- duction yields of baked breads with and without the addition of the culture filtrate and the commercial

,_,1.5

0.5

~ ' ' J i i i i i i i i

0 5 0 1 0 0 1 5 0

Culture period (h)

Fig. 5. Time course of acid production in the basal medium supplemented with 2.0% CSL and 2.0% DSP. Solid and broken lines mean acidities in the presence and absence of 3.58% bread crust, respectively.

Table 1. Amounts of total sugars and lactic acid in the basal medium supplemented with 2.0% CSL and 2.0% DSP

Amount (g/100 ml of culture broth)

Cultivation Fraction Total sugars Lactic acid D- L = DL-

Before Supernatant 1.27 _ 0.06 0.10 + 0.01 0.08 _ 0.00 0.18 ___ 0.01 Precipitate 1.19 _ 0.06 ND ND ND Tot al 2.46(100) 0.10 0.08 0.18

After Supernatant Precipitate Total

Difference in total amount

1.16 _ 0.17 0.63 _ 0.11 0.71 -t- 0.04 1.35 + 0.07 0.25 _ 0.02 ND ND ND

1.41 0.63 0.71 1.35 1.05(42.7) 0.53(21.5) 0.63(25.6) 1.17(47.6)

Percentages against the initial amount of total sugars are shown in parentheses.

Lactic acid fermentation

Table 2. Sensory evaluation for three types of baked bread

105

Sensory attribute Rank sums

+ Culture filtrate + Commercial fermented None seasoning

Flavor 20.0 ___ 2.1 23.3 + 2.6 28.7 + 0.0 Taste 16.7" + 1.0 28.3 _ 0.6 27.0 + 1.5 Overall acceptability 17.3" + 1.7 26.7 + 1.2 28.0 + 1.5

The rank positions (1 = first, 2 = second, 3 = third) for three breads given by 12 participants were added up to obtain rank sums in each sensory attribute. The lowest rank sum indicates the best liked one in the three breads. In these experiments, range of insignificant rank at the 5% level is from 18 to 30. *P<0.05.

seasoning. Table 2 shows the results of sensory evaluation performed by the ranking test method with respect to flavor, taste and overall acceptability. The rank sums of the taste and overall acceptability of the bread supplemented with the culture filtrate were lower than 18, which is the lowest insignificant rank sum at the 5% level, indicating this bread was significantly preferred over the bread made with the commercial seasoning.

filtrate can be applied to not only bread, but also other cereal products and may enable efficient recycle of bakery wastes.

ACKNOWLEDGEMENTS

We thank M. Awa, T. Yamakata and M. Yokota for technical assistance, and T. Kanetoki for critical reading of the manuscript.

DISCUSSION

Approximately half of the total lactic acid consumed in the world is produced by fermentation of carbo- hydrates by lactic acid bacteria. To supply the increasing demand for lactic acid, economical materials such as starch hydrolysate, whey and molasses have been evaluated to reduce the produc- tion costs (Peppier, 1977). Addition of YX is usually effective in improving the production rate and yield (Zhang and Cheryan, 1991; Montelongo et al., 1993). Good results were obtained in fermentation of whey ultrafiltrate supplemented with beet molas- ses, inoculated with a large volume of preculture medium composed of peptonized milk (Chiarini et al., 1992). In the present experiments, YX also stimulated acid production from bread crust most efficiently among the five compounds tested. The effect of CSL on acid production was indeed limited but enhanced by the simultaneous addition of DSP. Both CSL and DSP principally consist of nitrogen compounds, vitamins and minerals (Hoseney, 1990; Bell, 1989). Some nutrients lacking in CSL may be compensated by DSP and stimulate the growth of the lactic acid bacterium.

Lactic acid bacteria play an essential role in the production of sourdough breads (Sugihara, 1985). Sourdough bread is characterized by a typical flavor and better resistance to mold in comparison to non- sour bread (Salovaara, 1993). Since breadmaking by the sourdough method is complicated for industrial scale production, seasonings based on fermented milk, hops or sake mash, have been used to enhance the flavor and tastes by the Japanese bakeries. The present experiments show the culture filtrate derived from bread crust can be a substitute for commercial seasonings used in the breadmaking process. This

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