Screening of Bacterial Isolates Producing Diacetyl and Selecting the Most Efficient Isolate

Screening number of bacterial isolates in term of their ability to produce diacetyl from five samples of different dairy products include Qargoli cheese (Q and K) from two sources, Akkawi cheese (A), Soft cheese (S) and local butter (B), were purchased from the local market of Al-Ramadi city/Anbar province, the colonies were picked up from culture medium MRS and M17 after incubation 24 to 48 hr at 37C in anaerobic conditions and examined in microscopic (rod and cocci) Single, double and Strings, gram-positive and non-spore forming, we collected 7 colonies from 21 colony, 2 isolates from cheese (K) on M17 medium were cocci, 3 isolates from cheese (Q) on MRS medium were cocci, 1 isolate from cheese (A) on M17 medium was rod ales 1 isolate from cheese (S) but on MRS medium was rod, as for butter (B) no result, were shown under the same conditions, the 7 isolates subjected to a qualitative detection of their ability production and gave a positive indicator for Voges-proskauer test and Brady’s reagent to varying degrees between them, then the production of diacetyl for 4 isolates with the highest productivity, were quantified by Westerfeld method and HPLC technique, with Westerfeld the concentrations were 171.197, 234.654, 217.617, 222.926 μg/ml for K2, Q3, K4 and K7 respectively at absorbance 540 nm, isolate Q3 and K7 were distinguished with highest productivity of diacetyl by HPLC reached 5.7 and 5.9 ppm respectively.


Introduction
The traditional definition of LAB has been the formation of lactic acid as the main product from carbohydrate metabolism [1].LAB are a group of Gram-positive, non-spore forming, cocci or rods that excrete lactic acid as the main fermentation product into the medium if supplied with suitable carbohydrate [2].Diacetyl is a volatile taste component that naturally exists in many fruit and fermented foods, including blackberry, lucuma, yogurt, bread and vinegar, etc. [3].Some of the bacterial strains in supplementary cultures have the ability to encourage citrate metabolism and production of C4 chemicals such acetoin, diacetyl, and 2, 3-butanediol.For instance, during fermentation, Leuconostoc may create diacetyl, ethanol, and acetic acid, while Lactococcus lactis can produce diacetyl and acetoin.It has been demonstrated that Lactobacillus pentosus can create acetic acid, diacetyl, and ethanol effectively [4].Numerous microbes, including Streptococcus, Leuconstoc, Lactobacillus, Pediococcus, Oenococcus, and other microbial species, are capable of producing 1259 (2023) 012079 IOP Publishing doi:10.1088/1755-1315/1259/1/012079 2 diacetyl [5].In China, milk was fermented using leavening agents like Lactobacillus bulgaricus and Streptococcus thermophilus.The two strains could support each other's growth in culture and formed a symbiotic connection, Streptococcus thermophilus is typically regarded as the primary strain for the production of diacetyl and has a greater contribution to the flavour of fermented milk [6].Diacetyl, also known as butanedione (chemical name 2,3-butanedione), is a liquid that is yellow or light green in colour.Its molecular formula is an organic molecule with the formulas (CH 3 CO) 2 and C 4 H 6 O 2 [7].It has a strong butter smell and is readily soluble in ethanol, ether, propylene glycol, glycerol, and water when diluted to 1 mg/L [5].Diacetyl may provide a strong taste to the product at low concentrations since it has a low flavor threshold (1.5-5.0 g) [9].The reductive product, acetoin, has a much less aroma than diacetyl.There was natural diacetyl in Foeniculum vulgare, narcissus, tulip, raspberry and strawberry, lavender, citronella, rockrose, and cream [5].Diacetyl was primarily utilized in soft drinks, cold drinks, baked goods, candies, and other items as a flavour enhancer in the food sector.

Samples Preparation
Different samples of dairy products (five samples) were purchased from the local market in Ramadi, Anbar Governorate (Iraq), included Qargoli cheese from two sites, Akkawi cheese, soft cheese, and local butter, these dairy products were given the symbols K, Q, A, S, and B, respectively.

Isolation of Diacetyl-Producing Bacteria
By adding 1 ml or 1 g of each sample to 9 ml of sterile peptone water, decimal dilutions were performed, and the series of dilutions was finished up to 6-10 [10], after transferring 0.1 ml of each dilution to Petri plates in triplicate for each dilution, adding the solid nutrition medium MRS and M17 in the same way.The dishes were leaved some time to solidify, the samples were then put in an anaerobic jar, and were incubated at 37°C for 24-48 hr , then use a vacuum to remove all of the device's air.After that CO2 was added, and it was incubated under anaerobic conditions [11].

Screening of Isolates
 Cultural characterization: including color, shape, margin, elevation, texture, and size, was determined by Bergey's manual [12].Bacterial colonies growing single with phonotypical characteristics were collected on MRS Agar medium and M17 Agar medium, large or small, regular, flat, circular, with equal or uneven edges, smooth, non-sticky, white in color, while it was smooth, sticky on M17 Agar.They were plotted on the media on which they were grown, and this process was repeated for the purpose of purifying the isolates, Nature of growth on liquid media: The selected isolates were inoculated on MRS and m17 liquid culture media at 37°C for 48 hours, and their growth was monitored on the medium, Nature of growth on solid media: The properties of colonies growing on the surface of solid nutrient mediam MRS and M17 at 37°C for 48 hours were studied: shapes, sizes, appearance, colors and edges were studied. Microscopic characteristics: slides of isolate cells were prepared and stained with a gram stain and examined under a microscope(100x), in order to determine their response to this staining and to identify the shape of the cells and their aggregation and whether or not they form spores [13], using an oil lens of 100X magnification, the isolates were selected based on microscopic and cultural characteristics to determine the ability of it ' s to produce diacetyl compound via.

Qualitative Detection
Two methods were used for the qualitative detection of a diacetyl compound: the Voges-proskauer test and Brady's reagent.[14] This is a test for the production of acetylmethylcarbonil from glucose.To the inoculated medium after incubation, alkali is added, in the presence of which any acetylmethylcarbonil present becomes oxidized to diacetyl.The diacetyl will combine with arginine, creatine , to give a rose coloration.The medium used is glucose phosphate broth.After inoculation, incubate at the optimum growth temperature for 2-7 days.

Voges-Proskauer Test
 O'Meara's modification (O'Meara, 1931): add a drop (knife tip) of creatine to the culture followed by 5 ml of NaOH 40%.The tube was shaken well.The development of a pink colour in the centre indicates the presence of diacetyl and according to the intensity of the colour, usually within 30 minutes.
 Barritt's modification (Barritt , s 1936): 1 ml was taken from the culture and 0.5 ml of 6% αnaphthol solution and 0.5 ml of KOH 16% were added, the tube was shaken well, development of a red coloration, usually within 5 min, indicates the presence of diacetyl and according to the intensity of the colour.[15] 5 ml of the prepared reagent was added to a clean dry tube, to which 10 drops (2-3) ml of liquid culture were added, and the tube was shaken vigorously.The tube was gently heated in a water bath and waited for a few minutes, a precipitate appeared, paying attention to the colour of the precipitate.If it is yellow, it indicates the presence of an aliphatic compound, and if it is an orange colored precipitate, it indicates the presence of an aromatic compound, usually within 5 minutes.

Quantitative Estimate of Diacetyl
Two methods were used for the quantitative of a diacetyl ( Westerfeld 1945 method) and using HPLC technique.[16] 5 ml of the prepared test solution(cultural filter) after separating the cells from the filtrate by the centrifuge device and excluding the cells and adding 1 ml of Creatine 0.5% the prepared to the test solution and leaving for 10 minutes to allow the colour to change.Then 1 ml of α-naphthol 5% prepared is added and left for 30 minutes, after which the absorbance was read by a spectrophotometer at a wavelength 540 nm.Concentrations were calculated according to the standard curve of diacetyl (fig 2).

Standard Curve of Diacetyl
By adding different volumes of a standard diacetyl compound in test tubes, and completing the volume to 10 ml with distilled water, the absorbance of all tubes was measured using a spectrophotometer at a wavelength 540 nm(table 1) Only Blank's solution of distilled water was used to zero the device at the same wavelength.

Isolation of Diacetyl Producing Bacteria
Several colonies were obtained from the five local isolation sources on MRS and M17 medium, including (Qargoli cheese(K and Q), Akkawi cheese(A), soft cheese(S), and butter(B)).These colonies were then were grown under anaerobic conditions, in the presence of CO 2 gas, at a temperature of 37 °C, on suitable culture media (MRS Agar and M17 Agar).Colonies were re-grown (sub-culturing) more than once by the Streaking method under the same previous conditions in order to obtain pure isolates that are homogeneous in terms of color, size and shape.Accordingly, 21 isolates were obtained from the above isolation sources ( Table 2).While Karim obtained 60 bacterial isolates, 31 of which have the ability to degrade protein, 27 isolates that produce flavour compounds, including diacetyl, and 5 isolates that have the ability to degrade fats [18].

Cultural Characteristics
Studying the culture characteristics of isolated colonies by growing them on MRS and M17 Agar and incubating them at 37°C for 24 hours.21isolates were excluded from them, while the rest of the isolates were distinguished by being white in colour and others tilted to a creamy colour with equal edges and large in size.Some colonies were small in size, some of which are irregular in shape, sometimes they are convex in shape and have a non-sticky texture, while others have a sticky texture and grow at the bottom of the liquid medium MRS Broth (Table 3 ).Al-Janabi 1995 also determined the cultural and physiological characteristics of the colonies obtained through isolation of cheese and soil samples [19].Table 3. Cultural characteristics of the selected isolates.

Culture properties Characteristics of the selected isolates Colony appearance on solid medium
Large, regular, flat-shaped circular ones, and small circular ones, convex or flat-shaped edges of the colony With regular edges and some with uneven edges color White, glossy, and creamy in colour texture Smooth, not sticky and sticky The nature of growth on a liquid medium The growth is at the bottom of the medium because it has the ability to stick in the digestive system Al-Dosari 2002 also found that the nature of growth Lb.reuteri bacteria on MRS agar media are spindle-shaped, perpendicular to each other, creamy in colour, rough in appearance, while pointing to the bottom of the test tube when grown in MRS broth [20].

Microscopic Characteristics
The microscopic properties of the colonies after staining with Gram stain were studied under a light microscope.14 isolates were excluded from them ,they appear as yeast depending on the shape and size of the cell.The rest of the isolates were positive for Gram (rods, cocci), forming short chains, or in the form of single or double cells and non-spore-forming (Table 4).4. Microscopic characteristics of the selected isolates.

Microscopic properties Characteristics of the selected isolates Response to the gram stain
Gram positive Shape of the cells Rods , short or long and Cocci Rearranged Single or double and in short chains Spores non-spore-forming These results are consistent with the sources that indicated the main morphological and microscopic characteristics that characterize the genus Lactobacillus and Lactococcus, as they form circular, somewhat regular, sticky colonies with a white or creamy colour, positive for a gram stain, anaerobic, or need air in small quantities, and their ability to stick in the digestive system, so their growth is at the bottom of the liquid medium (MRS) [21].

Qualitative Detection of a Diacetyl Compound
The isolates with a sticky and spherical shape, G+ve and non-spore-forming were taken, and subjected to qualitative examination by Voges-proskauer and Brady ' s reagent as shown in table 5,6 and 7.Only 7 isolates were selected of a total 21 isolates according to agricultural and microscopic characteristic Table 5.The result of the Voges-proskauer test (O'Meara's Modification) appearance of a light pink colour.

No. Isolation symbol Appearance of a light pink colour
K +++ Table 6.The result of the Voges-proskauer test (Barrett's Modification) appearance of a purple colour.
No. Isolation symbol Appearance of a purple colour 5 shows the result of the Voges-proskauer test, the superiority of isolate 3Q ,7K and 4K by the appearance of light pink colour with three positive and two positive respectively, while the rest of the tested isolates gave a negative result.The same result was in table (6) for isolate 3Q and 7K with isolate 4K reaching (+++)signals by appearance of a purple colour.

Brady's Reagent
In the Brady's test (table 7) also shows the superiority of isolate 3Q and 7K with (+++) signals and lower than isolate 2K and 4K (++) signals .The results of the quantitative assessment of the diacetyl compound according to the above table showed that isolate K7 and Q3 gave the highest concentration diacetyl, followed by isolate K2 and K4, respectively, which are samples of Qargoli cheese.Based on the results of qualitative detection and quantitative assessment .Al-Sarraj 2005 obtained lactic acid-producing isolates using whey [22].Isolate 3Q and 7K were selected for the purpose of genetic diagnosis to know the genus and type of bacteria in another study.

Conclusion
Samples of different dairy products are useful in obtaining bacterial isolates that produce the diacetylflavor compound , which has other vital properties .Among the local dairy products Qargoli chees is the best source , from which bacteria were isolated with the highest productivity of diacetyl.So the medium M17 gave a higher number of colonies for lactic acid bacteria that produce diacetyl, We note that medium M17 gave an increase in the number of bacteria producing diacetyl compound, and this was proven by Yassin and et.al.When watermelon, grape, orange and pineapple juice was added to the whey, the number of bacteria increased after 18 hours of incubation, and the increase was clearer after 24 hours.The numbers were 119 x 107 , 133 x 107 , 108 x 107 and 125 x 107 c.f.u/ml.

2. 5 . 3 .
High-Performance Liquid Chromatography (HPLC)Diacetyl was estimated using HPLC technique[17], the separation conditions include the program for Diacetyl analysis by HPLC using an ODS C18 column (150 x 4.6 Id) mm, and a mobile phase consisting of (H 2 SO 4 ) 0.1%.At a flow rate of 1 ml/min, the reagent used is a UV-detector with a wavelength 192 nm, a retention time of (3.576)min.(fig3), and a runtime of 10 minutes.The sample was injected in a volume of 20 microliters manually and at room temperature.A filter was made for the models before injection.

Table 1 .
Absorbance readings and corresponding concentrations for the diacetyl compound.

Table 2 .
Isolated colonies from 5 local isolation sources and percentages of isolation.

Table 7 .
The result of the Brady's reagent test is brown crystals (granules).The result of (table 8) shows the superiority of isolate 3Q then 7K in the production of the diacetyl , as the percentage of each 234.654 and 222.926 µg/ml respectively.This agrees with the results of the qualitative detection in both types of test that were applied in this research.

Table 8 .
The concentration of diacetyl in the samples(Westerfeld) method.

Table 9 .
The concentrations of Diacetyl in the samples by the HPLC.Standard diacetyl retention time (3.576)min mentioned in figure 2.