Valorization of chicken viscera as natural raw material source: Application to hydrolysis of fatty esters for sewage treatment.

"Chicken viscera" constitute very abundant domestic wastes interestingly investigated in the present paper. The e�ciency of this crude slaughter co-product of high protein component, as biocatalyst, for the hydrolysis of fatty acid esters was reported and that, without any pre-treatment. The crude Chicken Intestines Powder (CIP) has shown a high reactivity for the hydrolysis of fatty esters. Two biocatalyst preparations were independently explored for the bioresolution of sec-phenyl alkyl carbinol esters: the CIP preparation and the crude Chicken Intestines Acetone Powder CIAP preparation. The last one has shown good catalytic activity during the bio-hydrolysis in biphasic medium. Furthermore, the direct hydrolysis of milk fat using CIAP (500 mg) reveals the elimination of fats present in 50 ml of treated milk. These results open up very interesting prospects for the use of this biowaste for the treatment of milk fat.


Introduction
Over the last thirty years, the worldwide supply of white meat has grown at an average rate of 5% per year [1].In 2017, this sector produced 100 million tons all over the world [2].The Food and Agriculture Organization of the United Nations (FAO) has reported that global chicken production has almost doubled over the last 25 years [3].In this context, and as a result of growing awareness of the environmental impact of e uent organic matter [4], the valorization of those waste for the elaboration of new natural raw materials has been a pressing priority for the scienti c community.Furthermore, the UN's sustainable development agenda has set vital targets to be achieved by 2030 to reducing wastes and protecting natural resources [5] including (i) the management and use of natural resources sustainably and e ciently; (ii) a signi cant reduction in the generation of waste through prevention, reduction, recycling, and reuse.
In Algeria, animal by-products are not recovered but disposed of [6], many of waste which are dumped or sold to informal sector recyclers who use them as animal feed [7].The National Waste Agency (AND) was estimated the industrial wastes in Algeria at 2.5 million tons/year, while the recovery rate did not exceed the threshold of 7%.Unfortunately, until now, this last area still untapped [8].Among the main objectives of sustainable development, reducing food wastes is a challenged task, either by avoiding its creation or by turning it into a resource.
The processing of live animal into a carcass generates noble products, essentially, muscle tissue compound (meat), as well as various co-products among: offal, bones, organic matter wastes, etc… [9].These biowastes are mainly buried in land lls and/or burned in incinerators, which contributes to environmental damage and disease transmission.
Chicken viscera constitute an important by-product of the meat industry.Owing to its composition of 92.5% organic matter, including 32.5% protein, 20% lipids, 40.1% carbohydrates, and 7.4% ash, seems a potential cheap biocatalyst candidate [20].
With the huge advancement of the industrial biotechnologies, the developments of new e cient and cheap biomaterials like biocatalysts still important.This is in order to develop sustainable, clean and viable alternatives to hard chemical processes [21,22].Industrial-scale organic biotransformations, including hydrolysis, oxidation, reduction, addition/elimination, and transesteri cation reactions were widely exploited in the pharmaceutical, chemical, food, cosmetics, and textile industries [23].Hydrolases, microorganisms or plants are also successfully used to access chiral building blocks through enzymatic catalysis [24][25][26][27].
The present paper reports for the rst time the valorization of a domestic biowastes "chicken viscera" as a potential biocatalyst source.Two preparation ways were described and used for the bio-hydrolysis of fatty esters, direct treatment of fat milk and the bioresolution of some racemic secondary esters of a high added value.
At the best of our knowledge, only the chicken liver acetone powder prepared in lab, was exploited for the resolution of racemic 1-phenylethanols and 1-phenylpropanols by the hydrolysis of their corresponding acetates [28] and for the hydrolysis of (+/-)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid esters [29].

Materials And Methods
Two biocatalyst preparations were explored from the whole chicken intestines.The crude Chicken Intestines Acetone Powder CIAP was used for the biohydrolysis of fatty acid esters and examined for the kinetic bioresolution of 1-phenyl ethanol.The e ciency of the last preparation was compared to the crude Chicken Intestines Powder CIP, during the bioresolution of several esters.

CIAP preparation procedure
For the rst preparation, the chicken intestines, freshly collected from a local slaughterhouse were cleaned and washed very carefully several times (Fig. 1).They were then subjected to grinding for 5 minutes in a blender.The shredded material was rinsed in acetone 3 times, ltered, and dried in an oven at a temperature of 35-40°C until a constant weight was attained.The dry mixture was then blended and the powder obtained, marked CIAP, was stored in a freezer at -4°C.From 1.5 kg of chicken viscera, 200 g of CIAP was obtained.The extract was tested in esteri cation and hydrolysis reactions.
The activity of CIAP preparation has been checked during the esteri cation and hydrolysis reactions (Scheme 1).It is to be underlined that only the biohydrolysis was checked with the liver acetone powder of animals, including chicken [30,31].The obtained results seem very interesting and differ according to substrate nature.Total hydrolysis of propyl laurate was recorded after 96h, whilst for the same reaction time the hydrolysis of glyceryl tris-butyrate did not exceed the threshold of 20% of yield.For the bioesteri cation of lauric acid by 1-propanol, the corresponding ester was recovered with 20% yield upon 7 days of stirring.These results showed a better e ciency of CIAP preparation for the hydrolysis of fatty acid esters.Which encouraged us to explore the e ciency of this biocatalyst for the treatment of FOGs (Fat, Oil and Grease) as a part of wastewater [32].For this purpose, we have envisaged to study its behavior directly on the milk fats.

Hydrolysis of milk fats
Milk fats present in dairy e uent and pose many problems for the water treatment.To explore the potentiality of our cheap biocatalyst preparation for the remediation of this problematic, we have examined its behavior directly on fresh milk fat.A primary chemical test [33], generally used to determine the fat content of the substance, often in milk and cream.This procedure based on the release of the fat matter from the mixture by appropriate acid treatment; it is then separated and collected in a graduated column by centrifugation.The CIAP preparation was added to 50 ml of fresh milk and stirred at 25°C for 24 h, then the mixture was ltered under vacuum, then the biocatalyst was recovered.The amounts of CIAP used were respectively: 100 mg, 200 mg, 300 mg, 400 mg, 500 mg and 1 g.The results were collected in Table 1.(b) Measured using the Geber method [33].
It was found that the fat present in the milk was completely eliminated for a quantity of 500 mg of CIAP (Fig. 2).These results have led to very interesting prospects for the use of this biowaste for the treatment of dairy water and could be extended to FOGs wastewater treatment.

CIAP catalyzed kinetic resolution of 1-phenyl ethanol
In the continuity of our laboratory investigations on the use of kinetic resolution reactions catalyzed by lipases to access to enantiomerically enriched building blocks, for example the fatty esters trough esteri cation and hydrolysis [34][35][36], we have scrutinized the enzymatic reactivity and selectivity of this newly developed biocatalyst.The kinetic resolution of sec-1-phenylethanol by acylation with succinic anhydride and by hydrolysis of the corresponding ester were selected [37][38][39].
Enzymatic acylation was performed on 1 mmol of 1-phenyl ethanol with 1 mmol of succinic anhydride and 250 mg of CIAP in 6 mL of Et 2 O for 4 days at 30°C.Hydrolysis of racemic acetate is performed on 1 mmol of 1-phenylethyl acetate with 250 mg of CIAP t in a mixture of 2 mL ethyl ether and 8 mL K 2 HPO 4 /Na 2 HPO 4 buffer solution pH = 7. Magnetic stirring was maintained for 4 days at 37°C.The resulting mixture of the product and the residual substrate was ltered over celite and extracted with ethyl ether (Scheme 2).
As described on scheme 2, the CIAP has shown a high reactivity for the hydrolysis without selectivity (C = 71%, E = 1) compared to the acylation one.These results con rm those obtained above with the achiral substrates.Thus reveal the hydrolytic properties of this biocatalyst preparation, for better apprehend the mode of action of this biocatalyst, we have extended the study of the biohydrolysis onto a set esters.We have focused on the impact of the leaving group on the reactivity of CIAP.Table 2.The results summarized in Table 2 showed that during the hydrolysis of esters 1e-1f, the hindrance of the ester group strongly impact the reactivity of CIAP, the conversion decreases from C = 71.4% to C = 51.6%, and then to C = 39% when going from the methyl derivative, to phenyl and to tertiary butyl respectively.A drastic decrease of the CIAP reactivity was noted when using decanoate derivatives as substrate (Table 2, entry 4: Conv = 10%) and total activity inhibition was recorded the laurate ester.This loss of activity is probably due to the di culty of reaching the active site of the enzyme when the size of the ester moiety increases.Changing the buffer solution K 2 HPO 4 /Na 2 HPO 4 by a Tris-HCl solution of pH = 8 where a slight advancement was observed Conv = 5.7% (Table 2, Entry 6), indeed, this solution has labile protons and facilitates the penetration of the substrate into the active site of the enzyme [40][41][42].

CIP preparation procedure
Following the obtained results we have envisaged to compare the rst preparation with the native chicken intestine powder, by omitting the acetone-extraction step (Fig. 3).The chicken intestines came from the same slaughterhouse (BENMERABT) [43], and were used directly after slaughter.They were thoroughly washed, cleaned, oven-dried at 50°C to constant weight, and then ground.The powder obtained is kept in the freezer at -4°C until it is used.From 1.5 kg of chicken viscera, 815 g of CIP was obtained.

Determination of the proteins content by the Lowry method
In order to establish the rate of advancement of the chemical reactions carried out with the two preparations, the amount of protein contained in each enzyme extract is assayed according to the method of Lowry et al [44].Protein concentrations were calculated by linear interpolation, using a standard range containing bovine serum albumin Fig. 4.
Using the calibration line, the protein concentration of the samples is calculated taking into account the dilutions used (Fig. 2).The results of the quantitative protein assay study by the Lowry method (Table 3) showed that both extracts have the same protein mass which is 25 mg/g.The results reported on Table 4 show that the selectivity of both preparations in the hydrolysis reaction for the majority of esters (1a, f-h) is variable 1 < E < 9 with conversions between 4% C 74%.Low selectivity (E < 9) was observed for derivatives 1a, 1f, 1g, and 1h upon the biohydrolysis.However, a higher hydrolytic activity of the CIAP compared to CIP was observed for these substrates, the conversion decreases from C = 71% to C = 26% for 1a (Table 4, entry 1 versus 2), from C = 65% to C = 53% for 1f (Table 4, entry 3 versus 4) and from C = 15% to total inactivity of CIP for the 1g ester (Table 4, entry 5 versus 6).CIP is active for the hydrolysis of 1h (C = 8%, E = 9) (Table 4, entry 8 versus 7) and selectivity of E = 9.Thus, the structure of the racemic esters signi cantly affects the reactivity and selectivity of both preparations.The shape and penetration of the substrates into the active site of the biocatalysts varied for both extracts and it was found that exhibited better catalytic activity than CIP even though the protein content of both preparations was similar.These results can be explained by taking into account the extraction procedure which probably impacts the shape of the active site in both extracts.

Conclusion
In the present work, for the rst time, a valorization of "chicken viscera" as a domestic organic waste of high protein content was described.The e ciency of two different biocatalyst preparations were scrutinized in the hydrolysis of fatty acid esters under sustainable conditions.The obtained results have revealed the hydrolytic propriety of this waste as potential raw animal material.Both preparations were very reactive in kinetic bioresolution of 1-phenylethyl esters via hydrolysis without selectivity.The CIAP preparation also allowed the removal of fat from fresh milk, which is particularly useful and valuable in the treatment of wastewater from dairies and oil mills.The exploitation of "chicken viscera" would reduce the negative impact of this waste on the environment, health, and development.Finally, all these results open interesting perspectives for the recovery of this waste and could constitute a new sustainable approach for the development of more integrated poultry production systems that contribute to reducing environmental damage.

Declarations Figures
Extraction procedure of CIAP Percentage of fat as a function of the amount of CIAP Extraction procedure for CIP

Table 1
Treatment of milk fat by CIAP.

Table 3
Protein determination of CIAP and CIP by the Lowry method.At this stage of our study, we have performed the hydrolysis on other types of racemic benzyl esters of potential interest with both CIPs.The reaction was performed in a biphasic medium (diethyl ether/buffer solution pH = 7: v/v: 2/4) in the presence of 250 mg of CIAP or CIP for one mmol of the substrate.The residual acetate and alcohol formed are recovered after 96 hours of reaction and separated on a silica gel chromatographic column.The enantiomeric excesses are determined by HPLC on a CHIRACEL OD-H chiral column.The results are shown in Table4.