Inexpensive α-amylase production and application for fiber splitting in leather processing

Abstract

Recently, the production of superior quality enzymes using waste sources has promoted greater research interest due to their enhanced enzyme activity, selectivity and stability. In this work, the production of enzyme α-amylase from wheat bran using a solid-state fermentation technique is presented. Further, reasonably high production of liquid α-amylase was achieved with enhanced activity and stability. In order to check the benefits of α-amylase, a fiber opening process using an in-house α-amylase has been developed for goatskins, in an attempt to reduce pollution from beam house processes, while doing a cost and environment benefit analysis. In addition, the effect of fiber opening by the developed enzyme was compared with a couple of commercial enzyme products and chemical processes (lime and sodium sulphide). After the fiber splitting process, the pelt was made into crust leather and the effect of fiber splitting on the strength properties related morphological changes of the crust leather samples were thoroughly investigated. The findings revealed that the inexpensive enzyme produced in this study displayed lower pollution load (COD, TS), with significant release of inter-fibrillary materials. Another significant observation was that the enzyme concentrate from the SSF process showed equivalent fiber splitting with lower cost than chemical-based processes and the commercially used powder enzyme products. Finally the developed inexpensive enzyme will act as a better replacement for chemical processes with lower cost.

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Introduction

Leather processing industries, especially those involved in conventional (chemical) pre-tanning or beam-house operations, have become synonymous with serious environmental pollution. Beam house processing, especially liming–reliming processes, are known to contribute 60–70% of the total pollution load in leather processing. In the new millennium, initiatives to address this through eco-benign approaches, especially enzymatic were taken and quite a few enzymes (proteases, lipases) for unhairing,¹ degreasing/defleshing² were developed and evaluated for basic properties as well as application at semi-commercial scales. This study involves fiber opening which removes inter-fibrillary materials (proteoglycans) by specific reaction of enzyme. An enzyme that has received lesser attention, nevertheless important, is α-amylase. First reference to the use of α-amylase skin connective tissue may probably be credited to Nishihara^3,4 who described the isolation of soluble collagen from ox-hide after pre-treatment of the minced tissue with α-amylase prior to extraction of the collagen. First use of α amylase for fiber opening of skins in leather processing was reported by Thanikaivelan et al.⁵ Among recent non-enzyme options, fiber opening of skins using ionic liquids has been demonstrated.⁶ Beam-house operations in leather processing, especially liming and reliming are categorised as critical processes because of the resulting high pollution load. Here, the hides/skins are subjected to cleaning before being prepared for subsequent tanning operation. Liming and reliming satisfy the objective of splitting up of fiber bundles making it easier for tanning agents, dyes, fat liquors, and other substances to diffuse into the matrix. In addition to that, the practice of liming claims to follow another step of deliming where ammonium salts are used for removal of lime which leads to liberation of toxic ammonia gas.⁷ Hence, fiber opening enzymes play a vital role in these treatments by replacing the hazardous chemicals involved in reliming/deliming. Fiber opening enzymes such as those available commercially are not necessarily labelled as such. They are generally bundled into other enzyme products such as those marketed for dehairing, bating or degreasing. The websites of some of the leading enzyme manufacturers Novozymes, Advanced Biochemicals, Noor Enzymes, Synkromax, Maps Enzymes, Tex Biosciences, Enzyme India, etc., present the list of enzyme products for leather processing. However, detailed features of most of these products are not readily available. Also, most often these products are spray dried formulations and contain some added chemicals as stabilizers and to impart other properties. As a result, the cost of the enzyme increases and in addition, the extra chemicals in these products could contribute to pollution load in the effluent. It is essential to study the effect of enzyme products having minimum of additives, it should be noted here that use of absolutely pure enzymes will be prohibitively expensive and not warranted for processes such as leather making. Reports claiming viability of integrated bioprocessing (dehairing and fiber opening) of leather have been based on work done with commercial powder enzymes.⁸ Hence, in this work, an attempt has been made to use an α-amylase product from a bacterial culture under solid state fermentation (SSF) using agro industrial residues for fiber opening in leather processing. The performance is compared with commercial enzyme products.

Materials and methods

Microorganism

A bacterial strain Bacillus subtilis MTCC 6537 previously isolated in the laboratory was shown to produce significant amount of α amylase in the culture medium.⁹ The organism was sub-cultured and maintained on LB agar at 4 °C. The culture on LB broth was used as inoculum for α amylase production by SSF. The capability of SSF for high yield and productivity of enzymes is well known.¹⁰

Solid state fermentation (SSF)

500 g of substrate mixture (75% wheat bran, 25% wheat rava) was taken, to this 25 ml of corn steep liquor was added and dispersed by mixing well. Then 500 ml of distilled water was added and mixed well. The moistened substrate was autoclaved at 1.1 kg cm⁻² pressure and at 121 °C temperature for 15 min and cooled to room temperature. Then the substrates were spread evenly on rectangular trays of approximate dimensions 430 mm × 230 mm × 60 mm, in 1 cm deep layers and inoculated with 24 h old inoculum and mixed well. The trays were then covered with aluminium foil. These operations were carried out aseptically in a laminar flow chamber. The trays were incubated at 30 °C for 72 h under static conditions. At the end of 72 h, the fermented material was transferred to a 5 l beaker and to this 1 : 10 ratio of 0.1 M phosphate buffer (pH 6.5) was added. The enzyme was extracted, centrifuged to remove biomass and analysed for amylase activity. Sodium benzoate (0.2% w/w) was added and the bulk cell free extract was stored at 4 °C. A portion of this extract was spray dried using a laboratory spray dryer (Labultima, Mumbai, India) after addition of maltodextrin (15% w/w), ammonium sulphate (0.5% w/w) and sodium chloride (2% w/w).¹¹ The concentrate was concentrated to about seven-fold using ultrafiltration unit (Pellicon, Millipore), which was diluted approximately to an activity of 125 U g⁻¹ for the application. The extract was pre-filtered via microfiltration and then subjected to ultrafiltration using a regenerated cellulose membrane with molecular weight cut off of 10 kDa. Both the liquid concentrate and spray dried amylase powder were taken for fiber opening application in leather processing. All the experiments were done in triplicates.

Amylase assay

The activity was determined by estimating the amount of glucose released as described by Miller with suitable modifications.¹² One unit of amylase activity is defined as the amount of enzyme that liberates 1 μmol of glucose per min under standard assay conditions. The enzyme activity is expressed in U g⁻¹ of the enzyme sample.

Evaluation of using enzyme on fiber opening/splitting application

Two soaked goatskins were dehaired using standardized dehairing process as developed earlier.¹³ For fiber opening evaluation, the methodology reported by Thanikaivelan et al.⁵ was followed. Each skin was cut along and across into four pieces and labelled as C, IH-A, IH-B, SH-A and SH-B. The wet weight of each skin after dehairing was noted. The control sample C was treated with 10% (w/w) lime and 300% water for 2 days followed by deliming and pickling. The experimental samples IH-A, IH-B, SH-A and SH-B were treated with 2% each of in-house amylase concentrate, in-house spray dried and two commercial enzyme products (supply house A and supply house B) respectively along with 100% water in a drum for 3 h, followed by pickling process.

Analysis of spent fiber opening liquor

The spent liquor from fiber opening process was collected from control and experimental samples and analysed for the release of proteins, carbohydrates, proteoglycans and glycosaminoglycans spectrophotometrically using standard procedures.^14–16 The spent liquor samples were also analyzed for the pollution parameters such as COD and TS using standard methods.¹⁷ The results are expressed in mg l⁻¹ of the sample.

Analysis of spent chrome liquor

Chrome tanning was done using conventional tanning procedure. The leathers after chrome tanning were piled for 24 h. The wet blue leathers were converted into crust leathers using conventional post-tanning method. The spent chrome liquor was analyzed for chromium content and the percentage uptake of chromium was calculated using standard procedures.¹⁸

Scanning electron microscopy

Samples from control and experimental skins after chrome tanning were cut from appropriate location.¹⁹ The samples were dehydrated using acetone and methanol and cut further into uniform thickness. Hitachi-SU6600 scanning electron microscope was used for the analysis. The micrographs for the cross-section were obtained by operating the SEM at low vacuum and an accelerating voltage of 5 kV with same magnification levels.

Physical characteristics of crust leather

The physical properties such as tear strength, tensile strength, elongation at break and grain crack for control and experimental crust leather were performed by standard procedures.^20–22

Results and discussion

Production of α amylase

It is well known that starch and starchy materials aid in the production of α amylase.²³ Wheat bran with a high content of starch (∼20%) is an excellent substrate for alpha amylase production. In this work, wheat bran was used for production of amylase by SSF using B. subtilis MTCC 6537 isolated earlier in our laboratory. SSF is a simple strategy for enzyme production in addition to being among the cheapest. Tray systems are quite simple and convenient to draw samples. Similar works in SSF have been reported Bacillus coagulans²⁴ and Bacillus sp. KR-8104.²⁵ Extraction of the enzyme from the bran followed by its concentration (seven-fold) yielded an activity of about 650 U g⁻¹ (diluted at the application site to get 125 U g⁻¹). Concentration of the extract after fermentation (72 h), stabilises its activity (Fig. 1) as well as simplifies the transport (due to reduction in bulk) to the user industry. The activity and related information of in-house concentrate, spray dried powder as well as commercial amylase powders of supply house SH-A and SH-B are described in Table 1. Differences in enzyme activity among various enzymes may be due to several factors, primarily microbial strain, type and amount of additives, etc. Since, assay methods differ from one manufacturer to another and generally not disclosed, it was necessary for all enzyme samples to be assayed by our method to establish a certain level of equivalence. It is also important to recognize here that it is common practice among enzyme manufacturers to formulate enzymes using additives viz., stabilizers, preservatives, viz., sodium benzoate, calcium chloride, potassium metabisulfite, dextrins, etc.²⁶

Fig. 1 Stability of crude and concentrated enzyme at 10 °C and RT (room temperature).

Table 1 Amylase activity of concentrate and powder samples^a

Samples	Form	Ingredients	Activity of 2% (w/w) amylase (U g^−1)	Fermentation
a IH-A: in-house concentrate, IH-B: in-house spray dried, SH-A: supply house A, SH-B: supply house B.
IH-A	Liquid concentrate	Enzyme + preservative	125 ± 15	SSF
IH-B	Powder	Enzyme + additives	175 ± 14	SSF
SH-A	Powder	Enzyme + additives	150 ± 10	SmF
SH-B	Powder	Enzyme + additives	205 ± 15	SmF

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Evaluation of using enzyme on fiber opening/splitting application

Conventional lime based fiber opening is a result of osmotic driven splitting while alpha amylase based fibre opening is due to the disintegration and subsequent removal of proteoglycans.⁸ In both cases, the net result is increased weight due to water absorption. The weights of the skins before and after fiber opening were noted. The percentage increase in weight after swelling was calculated from the difference in weights before and after fiber opening (Table 2). The percentage increase in weights of IH-A and IH-B were comparable with conventional lime control and commercial enzymatic treatment with SH-A and SH-B. Digital images (Fig. 2) taken for all the samples before and after treatment do not indicate any palpable fiber opening among the skins and hence, were subjected to SEM for better understanding.

Table 2 Change in weight due to chemical and enzymatic fiber opening^a

Samples	Before (g)	After (g)	Increase in weight (%)
a C-lime based conventional process.
C	350 ± 12	580 ± 15	66
IH-A	320 ± 14	540 ± 14	69
IH-B	310 ± 12	530 ± 12	71
SH-A	330 ± 13	540 ± 16	64
SH-B	370 ± 15	650 ± 14	72

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Fig. 2 The image shows the result of fiber opening of skin sample treated with different forms of enzyme. 1 – raw skin, 2 – before fiber opening and 3 – after fiber opening.

Analysis of spent fiber opening liquor

Fiber opening process removes the inter-fibrillary components, especially proteoglycans aiding the separation of the fibers and fibrils. Proteoglycans are extracellular proteins that are bound to glycosominoglycans with high molecular weight linear carbohydrates. The extent of fiber opening primarily depends on the removal of proteoglycans. Hence, the elimination of proteoglycans and other soluble proteins and saccharides (glycosaminoglycans) from skins can be used as a potential marker for quantifying the extent of fiber opening.²⁷ Madhan et al.²⁸ reported the effect of alpha amylase on inter-fibrillary materials including that 2% (w/w) was sufficient to optimally remove the latter as well matching the conventional lime based removal. The removal of inter-fibrillary materials after fiber opening for control and experimental is given in Table 3. A dose of 2% (w/w) of in-house enzyme (IH-A and IH-B) resulted in comparable removal of inter-fibrillary with that of the control as well as SH-A and SH-B.

Table 3 Analysis of spent fiber opening liquor

Samples	Proteins (mg g^−1)	Carbohydrates (mg g^−1)	Glycosaminoglycans (mg g^−1)	Proteoglycans (mg g^−1)
C	2.03 ± 0.02	1.89 ± 0.04	0.69 ± 0.01	2.32 ± 0.04
IH-A	2.70 ± 0.02	5.30 ± 0.01	1.20 ± 0.01	4.82 ± 0.03
IH-B	2.71 ± 0.03	4.98 ± 0.01	0.99 ± 0.02	4.85 ± 0.02
SH-A	2.64 ± 0.05	3.66 ± 0.04	0.96 ± 0.03	3.18 ± 0.01
SH-B	2.84 ± 0.03	6.28 ± 0.03	1.35 ± 0.01	4.89 ± 0.03

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The COD and TS values for control and experimental liquors are given in Table 4. The values for experimental leather processing are lower than control leather processing. The COD and TS loads for experimental liquor IH-A was much less compared to the control process. Conventional fiber opening processes are major contributors for the COD and TS in the spent liquor. This has been ameliorated using enzymes, which resulted in significant reduction in COD. The reduction in TS is mainly due to absence of lime in enzyme based processing.²⁹ Hence, the developed enzymatic process demonstrates significant reduction in COD and TS loads. In the case of SH-A and SH-B the levels were lower than chemical based process, similar to IH-B but higher than IH-A. The lower COD and TS values of IH-A compared to the other three enzyme products may be attributed to the absence of additives like starch derivatives, gum arabic, trehalose, etc. which are generally used for preparing powder enzyme products especially spray dried formulations.³⁰

Table 4 Pollution parameters

Process	COD (mg l^−1)	TS (mg l^−1)	kg t^−1 of raw skin		Relative levels (%)
			COD	TS	COD	TS
a Control (chemical process) taken as reference.
C	2512 ± 20	15 230 ± 45	24	145	100^a	100^a
IH-A	750 ± 10	1900 ± 20	6	14	25	10
IH-B	1546 ± 12	5210 ± 24	12	39	50	27
SH-A	1842 ± 16	6253 ± 31	14	47	58	32
SH-B	1732 ± 15	6524 ± 25	13	49	54	34

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Analysis of spent chrome liquor

Fiber opening or loosening of fiber bundles facilitates diffusion of the tanning agent and other post-tanning substances into the skin. Hence, chrome uptake is a standard measure of extent of opening up of fiber bundles.³¹ The chromium content in spent liquor for control and experimental samples is given in Table 5, which shows that the use of enzymes for fiber opening yielded similar chromium uptake as conventional liming method of leather processing.

Table 5 Spent chrome liquor

Sample	Cr2O3 content in liquor (%)	Uptake (%)
C	2.82 ± 0.13	65
IH-A	2.35 ± 0.12	71
IH-B	2.29 ± 0.07	71
SH-A	2.58 ± 0.09	68
SH-B	2.19 ± 0.10	73

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Scanning electron microscopy

The occurrence of fiber bundle splitting over the cross section of the chrome tanned leather was analysed by a SEM. From the detailed investigation of microstructure it can be inferred that, well dispersed fibers with more number of voids indicate fine splitting of fiber bundles in case of experimental leathers (Fig. 3). In general, the lime based fiber opening showed splitting of fiber bundles by osmotic pressure and the hydrostatic pressure built inside the matrix keeps the fibers apart from each other. The mechanism of the enzyme based fiber opening lies in disintegration of proteoglycans and its subsequent removal. A lower level of physical disruption in the enzyme-based system resulted in a cemented appearance in the experimental leathers treated with SH-A and SH-B. But the fibers are loose and well dispersed in experimental leathers due to the high level of proteoglycan cleavage obtained by enzymes and especially higher with IH-A and IH-B as seen by the individual fiber splits. The microphotographs of experimental leathers outlined the random distribution of the fiber, attributed to more splitting of the fiber bundles. It is thus inferred that, all the samples as well as control showed about the same degree of appearance/fiber opening.

Fig. 3 Scanning electron microscopic image showing the details of cross section of chrome tanned leather and the process of fibre opening by using (A) lime control, (B) IH-A, (C) IH-B, (D) SH-A and (E) SH-B.

Physical characteristics of crust leather

Tear and tensile strength tests were carried out for all the crust leathers at both along and across line. The grain crack strength for all the crust leathers was also evaluated. The values of the crust leathers corresponding to each experiment are given in Table 6. It is seen that both control and experimental leathers exhibit tensile, tear, grain crack and bursting strength values comparable to the BIS (Bureau of Indian Standards) norms.³²

Table 6 Physical characteristics of crust leather

Sample	Tear strength (N mm^−1)		Tensile strength (N mm^−2)		Elongation at break (%)		Grain crack
	Along	Across	Along	Across	Along	Across	Load (kg)	Distension (mm)
BIS norms	40–50	30–40	20–25	15–20	40–50	60–80	>20	>7
C	47.34 ± 4.16	38.29 ± 1.68	23.23 ± 0.89	16.63 ± 0.36	48.01 ± 2.13	75.01 ± 3.86	22 ± 2	9.12 ± 0.35
IH-A	45.37 ± 2.49	37.87 ± 1.75	21.52 ± 1.12	16.92 ± 0.53	48.67 ± 3.11	74.14 ± 4.23	21 ± 3	9.04 ± 0.42
IH-B	42.72 ± 3.01	35.74 ± 2.11	23.49 ± 1.23	15.95 ± 1.02	48.54 ± 2.94	76.37 ± 3.67	22 ± 2	8.99 ± 0.59
SH-A	40.25 ± 2.13	35.56 ± 2.25	21.07 ± 0.95	15.75 ± 0.69	46.67 ± 2.82	79.17 ± 4.92	20 ± 2	7.88 ± 1.65
SH-B	43.82 ± 3.46	36.99 ± 1.98	22.90 ± 1.09	16.19 ± 0.52	46.75 ± 2.64	75.52 ± 3.42	24 ± 4	8.98 ± 1.05

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Cost benefit analysis

The main impediments in acceptability of enzymes for replacing chemicals is the consistency in composition and stability, relatively higher cost, modifications in process handling systems, etc. However, it is mainly the cost factor that is considered to outweigh the others. For chemical and enzyme based processing of 1 ton of soaked goatskins, a preliminary cost assessment has been done (Table 7). Two of the ingredients are common to all the trials viz., ‘in-house’ dehairing protease and basic chromium sulphate (BCS). As for both the commercial amylases, a cost of USD 30.7 per ton of skins were taken. The cost of in-house amylase powder (formulated using similar additives that are practiced in industry) is also assumed as USD 30.7 per ton of skins for simplified comparison. From our earlier report, the estimated cost of concentrate is about USD 2.5 per liter³³ and cost for the application is hence, USD 10.5 per ton of raw skins. Thus, it is seen that the use of amylase concentrate for fiber opening processing costs lower than that of spray dried and commercial enzymes as well as chemical based process. The reason that the market enzymes cost slightly more is not only because of chemical additives added during spray drying but also due to the practice of reformulating enzymes acquired from other sources (imports, etc.) which invariably spike the cost. In this study, to simulate the commercial formulation, maltodextrin, ammonium sulphate and sodium chloride were added in order to obtain a spray dried product and the cost was hence, taken as the same as those of the supply houses. In this work, the lower cost of the amylase concentrate is due to use of SSF which is inherently a low cost technology, with a much higher productivity compared to liquid fermentation and devoid of additives except for a preservative, sodium benzoate (0.2% w/w). Thus, this form of the enzyme turned out to be the cheapest. The overall leather quality is quite comparable with chemical based process.

Table 7 Cost benefit

Chemicals/bioproducts	C (US $ per t of raw skins)	IH-A	IH-B	SH-A	SH-B
In-house dehairing protease	25.9	25.9	25.9	25.9	25.9
Reliming	7.7	—	—	—	—
In-house amylase	—	10.5	30.7	—	—
Supply house A	—	—	—	30.7	—
Supply house B	—	—	—	—	30.7
Ammonium chloride	11.5	—	—	—	—
Sodium chloride	7.7	7.7	7.7	7.7	7.7
Sulphuric acid	1.4	1.05	1.05	1.13	1.20
Basic chromium sulphate	98.7	98.7	98.7	98.7	98.7
Sodium formate	15.4	15.4	15.4	15.4	15.4
Sodium bicarbonate	2.7	2.7	2.7	2.7	2.7
Total	171.0	161.95	181.85	181.93	182.0

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Conclusions

Several enzymatic bioprocessing strategies have been reported in the last 15 years, nevertheless their translation to industrial practice is hardly significant. There is a general reluctance on the part of the tanners to adopt enzymes mainly due to their higher cost and hence, it is imperative to find methods to reduce the cost. The strategy of using amylase concentrate has great potential to reduce the cost and eliminate pollution in the environment as indicated by the lowest COD and TS levels in this work. The extent of fiber opening achieved in leather processing using amylase was comparable with that of conventional lime based processing and other commercial enzymes. The analysis of pollution parameters shows that the enzymatic process contributes to a significant reduction of COD and TS compared to conventional processing and bioprocessing using commercial enzymes, with an added benefit of elimination of the deliming step. The application of the concentrate directly over skins appears to be technically feasible. The main aim of this work was to minimize the chemical content in the enzyme product taken for bioprocessing. The study was conducted with goat skins only and an extended study using other skins and hides would be required for a general understanding of this enzyme process. The use of this in-house enzyme concentrate is the first of its kind and has yielded crust leather of acceptable quality, a cleaner effluent and most importantly an affordable alternative compared to control as well as commercial enzymes. Besides, the fact that the production of enzymes by solid state fermentation is viable at small scales, great potential exists for those tanners who would like to patronize on-site production of enzymes for consistent product quality and better process control. In this study, the commercial enzymes were arbitrarily chosen by us for the research work. The results of this study are based on tests performed under certain conditions only and are in no way intended to reflect on the overall quality aspects of these enzyme products as a whole. A more thorough comparison would warrant the use of all available commercial enzyme products and conducting investigations under different conditions and raw materials viz., sheep skins, cow and buffalo hides.

Acknowledgements

The authors thank Director, CLRI, Chennai, India, for kind permission to publish this work. Financial support received for carrying out this work from CSIR STRAIT (WP-1.2.7 of CSC 0201) is gratefully acknowledged. Sincere thanks are due to Mr S. Prasanna, Leather Processing Division for Technical assistance.

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