THIS INVENTION relates to the identification and characterisation of probiotic bacteria isolated from bovine tissue and the ability of the bacteria to colonize human gut cells to synthesize vitamin A in the form of retinol and b-carotene.

Background of the invention

One of the current millennium development goals targets “Children, Food Security, and Nutrition” is the prevention of diseases from childhood blindness to mortality due to vitamin A deficiency disorders (VADD). VADD is associated with serious health issues such as frailty, visual impairment, and increased risk of infant morbidity and mortality, especially in individuals under five years of age and in pregnant women. UNICEF reported that in 2018 the highest rates of vulnerable children are in Sub- Saharan Africa (48%) and South Asia (44%). Pseudo vitamin compounds are used in fortification programs to reduce morbidity due to VADD, but they are associated with adverse side effects and hypervitaminosis. There is, therefore, a need for viable and natural alternative strategies to combat VADD.

Vitamin A is present in the body as b-carotene and retinol. It is an appealing consideration to use gut-friendly bacteria to produce vitamins through viable natural strategies. Probiotics are live microorganisms that have associated health benefits when consumed or applied to the body. The inventors believe that identifying suitable gut-friendly bacterial strains would lead to the development of a probiotic product that is capable of producing desirable levels of vitamin A in order to alleviate vitamin A deficiency and promote good health. The use of probiotic bacteria for in situ vitamin fortification offers an economic strategy that is less likely to have side effects due to elevated concentrations of vitamins.

In the last two decades, researchers have reported on the production of b-carotene and/or other carotenoids by bacteria including Lactobacillus plantarum, Micrococcus sp., Sphingomonas jasps\, and Flavobacterium multivorum. Different probiotic vitamin- producing bacteria have been studied and are available in the probiotic market; however, gut-friendly bacteria that produce b-carotene and/or retinol remain to be identified and have not been or are scarcely reported so far. Likewise, genetically engineered bacteria with b-carotene producing capacity that have been developed have been unsuccessful due to their unintended adverse side effects.

Retinyl palmitate (preformed vitamin A) and b-carotene compounds are primarily found in animal tissues, including the bovine liver. Animals cannot produce retinoids de novo. The bovine liver is the top animal source of vitamin A (4 - 20 mg retinol/ 100g) which can store and maintain adequate levels of retinol and b-carotene for months to years. Cows rely on pro-vitamins like carotenoids from plant-based fodders (60 - 120 mg b-carotene per 100 g). A biochemical conversion takes place in the bovine intestine and liver where b-carotene is cleaved into vitamin A through b- carotene-15, 15’ mono(di)oxygenase (BCMDO)-like enzymes. Higher BCMDO enzyme activity has been identified in bovine liver and intestinal tissues compared to brain, lung, and kidney. Bacteria colonized in the liver play significant roles in the absorption and metabolism of vitamin compounds.

Certain strains of Staphylococcus, Streptococcus, and Bacillus isolated from animal tissues are known to synthesize carotenoids. Variations have been observed in liver storage of b-carotene and retinoids in response to the alteration in the rumen microbial organisms. The gut microflora plays a role in the bioavailability of carotenoids, and an indirect mechanism may be responsible for this.

These organisms have the potential to act as cellular carriers and to deliver vitamin A- like bioactive compounds in human gut cells. It has been recognised that probiotic bacteria with the ability to produce and/or modulate vitamin A metabolism should be mined and utilized holistically to manage vitamin A deficiency. However, no attempts have been made to determine the capacity of bacteria associated with the liver, intestine and rumen of green forage-fed or free-range cattle in the biosynthesis of b- carotene and/or retinoids.

Here, we have isolated probiotic bacteria from bovine tissue and identified the bacteria’s ability to synthesize vitamin A compounds (b-carotene and/or retinol), and their probiotic potential and gut-friendly properties have been characterised. Lactobacillus plantarum is a gram-positive lactic acid bacterium commonly found in fermented food and the gastrointestinal tract and is commonly used in the food industry as a potential starter probiotic. Among the lactic acid bacteria, L. plantarum has attracted a lot of research because of its wide applications in the medical field with antioxidant, anticancer, anti-inflammatory, antiproliferative, anti-obesity, and anti diabetic properties. Here, L. plantarum probiotic strains isolated from bovine organs have been identified as having the property of producing vitamin A, in the form of b- carotene and retinol. These strains are DSM 20246, DSM 16365T, DSM 2601 and DSM 12028. L. plantarum DSM 16365T is shown to have a high b-carotene yield and shows essential probiotic properties that are suitable for food biofortification. The Escherichia coli strain DSM 682 is also identified as probiotic bacteria that synthesize high levels of vitamin A.

Summary of the invention

According to the first aspect of the invention, there is provided probiotic bacteria isolated from animal tissue for the synthesis of vitamin A. The bacteria is from a bacterial strain that may be isolated from bovine tissue, in particular liver, intestine, and rumen tissues.

Vitamin A may be synthesized as retinol and/or b-carotene.

By “strain” is meant, a member of a bacterial species with a genetic signature that differentiates it from other members of the same bacterial species.

By “isolated” is meant that the bacteria have been separated as a pure culture from at least some of the components of bovine organs (liver, intestine, and rumen), where the bacteria are naturally colonized. The bacterial strain may be selected from Lactobacillus plantarum bacterial strains which may include DSM 20246, DSM 16365T, DSM 2601 , and DSM 12028 isolated from bovine liver and Escherichia coli strain DSM 682 isolated from the bovine intestine. The bacteria synthesize vitamin A under aerobic and anaerobic conditions.

According to a second aspect of the invention, there is provided a composition comprising at least one probiotic bacterial strain isolated from bovine liver and intestinal tissue for the synthesis of vitamin A in the form of retinol and/or b-carotene.

The bacterial strain may be selected from Lactobacillus plantarum bacterial strains including DSM 20246, DSM 16365T, DSM 2601 and DSM 12028, and Escherichia coli DSM 682.

The composition may be in the form of a synbiotic food.

The probiotic bacteria isolated from bovine liver and intestinal tissues remain substantially stable in gastric juice of a subject.

The probiotic bacteria may have the ability to colonize human gut epithelial cells to synthesize vitamin A.

The probiotic bacteria may have essential probiotic properties suitable for food biofortification.

According to a third aspect of the invention, there is provided the composition according to the second aspect of the invention for use in a method of increasing vitamin A levels in a subject with low levels of vitamin A by colonizing with the subject’s gut cells to synthesize vitamin A in the subject in the form of retinol and/or b-carotene.

According to a fourth aspect of the invention, there is provided a use of isolated probiotic bacteria from bovine liver or bovine intestinal tissue in the manufacture of a composition for treating a subject with low levels of vitamin A, wherein the bacteria colonize with the subject’s gut cells to produce vitamin A in the subject in the form of retinol and/or b-carotene. The bacteria may be from a bacterial strain selected from a Lactobacillus plantarum bacterial strain including DSM 20246, DSM 16365T, DSM 2601 and DSM 12028, and an Escherichia coli strain DSM 682.

The composition may be in the form of a synbiotic food.

The invention will now be described in more detail with reference to the Example hereunder, and the accompanying drawings.

Brief description of drawings

In the drawings,

FIGURE 1 shows, for the Example, b-carotene and retinol production by bovine bacterial isolates, (A) with supplementation of 2% glycerol, (B) in aerobic and anaerobic incubation in the selective broth medium, the bacterial strains with higher vitamin A compounds production are highlighted with asterisk marks and values with different superscript letters differ significantly (P< 0.05);

FIGURE 2 shows, for the Example, time-of-flight mass spectrometry chromatogram (0-14 min) of b-carotene and retinol produced by bovine isolates, (A) chromatogram of L. plantarum DSM 20246, (B) chromatogram of L. plantarum ssp. argentoratensis DSM 16365T, (C) chromatogram of L. plantarum DSM 2601 , (D) chromatogram of L plantarum DSM 12028, (E) chromatogram of E. coli DSM 682, and (F) chromatogram of L. acidophilus ATCC 4356;

FIGURE 3 shows, for the Example, tolerance of vitamin A producing bovine bacteria to different environmental stresses - (A) temperature (4 °C, 10 °C, 37 °C and 45 °C); (B) ethanol (0%, 5%, 10%, and 15%); (C) NaCI (0%, 2%, 5%, and 10%); and (D) 0.3% bile salt (1 h, 2 h, 3 h and 4 h); bacterial growth is evaluated through OD values at 600nm after 24h of incubation, values with different superscript letters differ significantly (P< 0.05); and

FIGURE 4 shows, for the Example, Biplot (axes F1 and F2: 86.53%) of principal component analysis visualizing the growth of the bovine strains at temperature (10 and 45 °C), ethanol concentrations (5 and 15% ethanol), bile salt (0.3%), NaCI (5 and 10%) and b-carotene and retinol production at aerobic and anaerobic conditions. EXAMPLE

Materials and Methods

Isolation and identification of probiotic strains from bovine organs and their growth conditions

Bovine liver, rumen and intestine tissues (n=3) from grass-fed or free-range cows were obtained at an abattoir in Gauteng, South Africa. All the tissue samples were harvested within 30 minutes after slaughter and stored in a cold room at -20 °C until required for the assay. The commensal probiotic bacteria were isolated from the bovine tissue samples as described below. For all samples, 10 g of tissue sample was removed aseptically from the bovine organs and added to 40 ml of 0.1% peptone buffered saline water (Merck, South Africa) in a sterile stomacher bag and stomached for 5 min at 230 revolutions per minutes in a Stomacher lab blender-400 (Seward Ltd., London, UK). In the next step, 10 ml of the bovine bacterial suspension of each organ (n=3) was added to 40 ml of de Man, Rogosa, Sharpe (MRS) broth, nutrient broth (Oxoid, Basingstoke, UK) and Luria and Bertani broth (LB) broth (Oxoid) separately and homogenized by vortex mixing. The inoculated broth samples were incubated at 37 °C for 24 h under aerobic and anaerobic conditions (Jaglan et at., 2019). Tubes showing turbidity were selected and plated onto MRS agar, Violet red bile agar, Nutrient agar and Slanetz and Bartley agar plates and incubated as described above. Colonies with distinct morphologies were purified and stored in 25% glycerol at -20 °C until further use. The identification and classification confirmation of bacterial isolates was carried out in Matrix-assisted laser desorption/ionization coupled with time-of-flight mass spectrometer (MALDI-TOF MS) (Bruker Daltonik GmbFI, Germany) equipment. After 24 to 48-hour cultivation, the single colony cultures that were spotted on the selective media were transferred with a toothpick onto a steel MALDI Biotyper target plate (MSP 96 polished-steel target) (Bruker Daltonik GmbH, Germany). The plates were air-dried, and the spots were overlaid with 1 pi a-cyano-4-hydroxycinnamic acid solution (Bruker HCCA matrix portioned, Art. #255344).

The mass spectra of bacterial culture were generated with mass spectrometer operated by the MALDI Biotyper automation control and recorded by FlexControl software (Bruker Daltonics, Bremen, Germany). The spectral peak list was used for matches against the reference spectra library using an integrated pattern-matching algorithm of the Biotyper 3.0 software (Bruker Daltonics, Germany). The best matching database records were generated by logarithmic identification score ranging from 0 to 3 levels, whereas the score < 1.700 indicate poor or no identification, 1.700-1.999 indicate genus identification, > 2.000 indicate species identification and >2.300 indicate species identification with a high level of confidence (Cherkaoui etal., 2010). b -carotene and retinol production bv bovine isolates

The biosynthesis of b-carotene and retinol was screened through the microbiological assay method suggested by Miller et al. (2013). Individual colonies from each of the bovine probiotic bacterial plates were inoculated into 10 ml of the appropriate broth and aerobically incubated at 37 °C with shaking at 150 rpm overnight. These overnight cultures at Oϋboo 0.01 were diluted further into fresh media and incubated at 37 °C with shaking at 150 rpm. Lactobacillus acidophilus ATCC 4356, Bacillus subtilis ATCC 168 and Escherichia coli ATCC 8739 were included as controls to compare the vitamin A production capacity with bovine bacterial isolates.

A similar procedure was adopted for b-carotene and retinol production and quantification assay. The bacterial cultures grown overnight, as described above, were inoculated into the appropriate broth of 10 ml to achieve a starting Oϋboo value of 0.01. All tubes were wrapped with alumina foil and or brown bottle during incubation to limit exposure to light in a rotary shaker at 150 rpm and 37 °C. After 24 h of incubation, the cells were centrifuged at 3000 rpm for 15 minutes, and the cell pellets were extracted with tetrahydrofuran (THF) (Miller et at., 2013). The spectrophotometric assay was conducted to quantify the vitamin A compounds in the bovine bacterial extract using T80+ UV/Vis Spectrophotometer (PG Instruments Ltd., United Kingdom). The absorbance was recorded at 460 nm for b-carotene and 325 nm for retinol. The standard curves were obtained by preparing a serial of standards in THF.

Effect of culture conditions and glycerol supplementation on vitamin A production

The effect of growth conditions such as aerobic/ anaerobic and glycerol supplementation (0.5, 1 .0 and 2.0%) on the bacterial production of b-carotene and retinol was investigated. The cultures were incubated in dark and anaerobic condition using AnaeroGen™ 3.5 L sachets (Oxoid Ltd., Basingstoke, UK) in 3.5-litre jars for 24 h at 37 °C with continuous shaking at 250 rpm. The addition of the extra carbon source like glycerol has been reported to increase the production of pro-vitamin compound b- carotene (Yoon et at. 2007). UV spectroscopy was used to determine the vitamin A compounds, as discussed above.

Extraction and determination of vitamin A compounds through UHPLC-MS/MS analysis

The presence of b-carotene and retinol in cultural pellets of the bovine bacterial isolates were further confirmed and quantified through UHPLC-MS/MS with little modification of the method suggested by Plozza et al. (2012). All-trans b-carotene, retinol and retinyl acetate were purchased as standard substances from Sigma-Aldrich (USA). Acetonitrile, chloroform, and tert-butyl hydroxyl toluene (BHT) of first grade were purchased from Merck Millipore (USA). The stock solutions of retinol and retinyl acetate were prepared in ethanol and b-carotene was prepared in chloroform with 0.05 % BHT and stored in amber bottles at -20 °C. The working standard solution was prepared with the mobile phase (80% Acetonitrile: 20% ultrapure water) every time with 0.05 % BHT. Since fat-soluble vitamins are light-sensitive, precautions were taken to minimize exposure of the extracted solutions to daylight by using either amber glassware or ordinary glassware protected with aluminium foil.

The b-carotene and retinol were extracted as above, and the THF extract was dried by nitrogen flushing. The residue was dissolved in 1 ml of the UHPLC mobile phase. The UHPLC-MS/MS analysis was carried out using the Waters Acquity UHPLC ^® hyphenated to a quadrupole-time-of-flight (QTOF) mass spectrometry system. The system was operated with MassLynxTM (version 4.1 ) software and Waters ^® Synapt G2 high definition mass spectrometry (HDMS) system (Waters Inc., Milford, Massachusetts, USA). For each target compound, the accurate mass molecular ions (M+H) + (b-carotene - m/z 535.43, retinol - m/z 269.22 and retinyl acetate 269.23) (Hinchliffe et al. 2016) were selected as the quantification ions for peak area calculation. The retention widow criterion was set at 0.2 min, and the mass window criterion was set at 0.02 Da. The separation was completed using a reverse-phase step gradient elution scheme from 50% ultrapure water to 100% acetonitrile with 0.05% BHT. The column temperature was kept constant at 40 °C, and the flow rate was set at 0.5 mL/min for the entire run giving a total run time of 15 min.

The calibration was an internal calibration method with preparation of two series of standard preparations with different molar ratios of retinol - retinyl acetate and b- carotene - retinyl acetate (0.05:1 , 0.1 :1 , 0.5:1 , 1 :1 , 5:1 , 10:1 , 50:1 , 100:1 and 500:1). The internal standard solution was prepared at 1 pg/ml. The accuracy of the method was evaluated with internal standard recovery, which was added to the bacterial broth at the beginning of extraction. Earlier, the mobile phase solvent was run to confirm the absence of any analyte compounds.

Tolerance to environmental stress, gastric juice and bile salt

The bovine bacterial strains with vitamin A producing capacity were characterised for probiotic characteristics. The Lactobacillus strains were inoculated in MRS broth at 37 °C for 24 h, and 0.1 mL was transferred into 5 mL MRS broth, and E. coli into LB broth. Cells were centrifuged (5000 rpm, 5 min), washed and suspended in 5 ml of phosphate-buffered saline (PBS) (pH 7.0) and applied for subsequent assays such as tolerance to temperature, pH, gastric juice, salt, ethanol and bile salts (Li etal. 2017). The total viable bacterial cell count of the washed cell suspension was determined prior to the assays. Concerning the environmental stress tolerance assay, the bacterial growth was observed at different levels of incubation temperatures (4, 10, 37 and 45 °C) and 0.3 % bile salt (0, 1 , 2 and 4h) and different concentrations of salt (NaCI) (0, 5, 10 and 15%) and ethanol (0, 2, 5 and 10 %). The bacterial growth was evaluated by measuring OD6oo nm after 24 h of anaerobic incubation.

Further, the tolerance to gastric juice was studied with 0.3 % (w/v) of pepsin (Sigma- Aldrich, USA) in PBS buffer, with pH 2.0, 3.0 and 4.0, respectively. Around 200 mI of cell suspension was poured in 5 ml of PBS along with 300 mI of NaCI (0.5 % w/v) and 1 ml of simulated gastric juice. After incubation at 37 °C for 0 h, 2 h and 4 h, an aliquot of 100 mI was removed and plated on MRS media. The viable count of the stains was determined anaerobically after 48 h of incubation and expressed as log 10 CFU/ml.

Statistical analysis

The statistical tests were conducted using Microsoft Excel Spreadsheet (Microsoft, USA) and SPSS version 25 package (USA). Analysis of variance and student’s t-test was used for comparison of means. The interactions among the pH, time and bacterial counts (log CFUs) were analyzed using multivariate ANOVA repeated measure option in SPSS. The biochemical and probiotic characteristic data obtained were analyzed using principal component analysis (PCA). All data are representative of at least three independent experiments, and the values were expressed as the mean ± standard deviation. Significant difference among the means was set at ‘P< 0.05’.

Results and Discussion

Isolation and identification of bovine bacteria through MALDI-TOF MS analysis Table 1. List of bacterial isolates from cow liver, intestine and rumen tissues identified and confirmed through MALDI-TOF MS ¹

¹ ATCC cultures of Lactobacillus acidophilus, Bacillus substillis and Escherichia coli were taken as control samples and were correctly identified. All the bacterial isolates were identified with > 2.00 MALDI-TOF score.

The results obtained through MALDI-TOF MS analysis (Table 1) enable reliable identification of 34% of Lactobacillus genus, 24% of Enterococcus genus and 42% of Escherichia and Bacillus genus from the bovine organs. 25 out of 33 bovine bacterial isolates display >2.3 MALDI-TOF mass spectra score values and are identified to the species level with a high level of confidence. The reference strains Lactobacillus acidophilus ATCC 4356, Escherichia coli ATCC 8739 and Bacillus substillis ATCC 168 were used to validate the in-house database. Not surprising, all the isolates from bovine rumen, liver and intestine are facultative anaerobe or obligate anaerobes in nature. The bovine isolates display better capabilities in sugar fermentation and glycerol utilization in comparison to ATCC control cultures (data not shown here) because they are originally from a gut environment. The identification of facultative anaerobic bacteria through MALDI-TOF MS is authenticated by many researchers because of its high power in species-level taxonomic resolution based on the ribosomal and housekeeping proteins. b -carotene and retinol production of the bovine bacterial strains The tetrahydrofuran extracts of b-carotene are detected at its characteristic absorption maximum near 460 nm and retinol at 325 nm through UV spectroscopy. The selective broth for the Lactobacillus (MRS) and Escherichia (LB) was chosen as a production medium to compare the strains with ATCC controls which are negative for b-carotene and or retinol production. Eight isolates were shortlisted from the total isolates with higher b-carotene production (more than 10 pg/g dry cell weight) capacity after five rounds of transfer and are defined as b-carotene producers. An effective range of b- carotene (19.25±0.55 to 89.05±1 .95 pg/g dry cell weight) is consistently observed after aerobic incubation in multiple independent experiments (Figure 1). Several authors support the bacterial production of vitamin compounds in the bovine rumen environment. The ruminants acquire vitamins through symbiotic bacteria that are colonized on the four chambers of the rumen, which is absorbed in the intestine and stored in the liver. The carotenoid intake by the ruminants is absorbed into liver and intestine, where the enzyme BCM(D)0 and other colonized microbes cleave it into retinol molecule. Those bacteria may have acquired those genes through horizontal gene transfer, hologenomic adaptational effect, a bi-directional pressure or putative enzymes. In the determination of extracellular vitamin A compounds, the cultural supernatant was assayed in UV spectroscopy, and an insignificant absorbance is found which is reduced to UV spectral background interferences in comparison with the blank samples (data not shown here).

Effect of carbon source and incubation condition on the production of vitamin A compounds

To generate b-carotene and retinol in the human gut environment and to evaluate the biofortification feasibilities, the bacterial growth and vitamin A production in anaerobic condition and supplementation of glycerol as a carbon source is evaluated. In the glycerol supplementation experiment, higher b-carotene production is found with 2.0 % glycerol (Figure 1A). The efficiency of the b-carotene output with glycerol as a carbon source is experimental to maximize isopentenyl diphosphate biosynthesis in the mevalonate pathway. The anaerobically grown cultures also show increased production of vitamin A compounds (Figure 1B). Importantly, four of the L. plantarum (DSM 20246, DSM 16365T, DSM 2601 , DSM 12028) strains and one E. coli (DSM 682) strain produce higher b-carotene among the examined strains with the addition of the carbon source (25.69±1.20 to 198.01 ±3.28 pg/g dry cell weight) and in an anaerobic condition (26.58±2.58 to 110.25±4.12 pg/g dry cell weight). The retinol production does not differ with supplementation of a carbon source. These results are in line with the finding that the b-carotene biosynthesis pathway is active due to a higher glycolysis pathway flux when there is the optimum temperature, incubation environment and carbon source for the bacteria. The potential to survive in aerobic and anaerobic environments and the ability to metabolize different carbon sources are attributes of probiotic bacteria to develop synbiotic foods with biofortification approaches.

UHPLC-MS/MS analysis of b-carotene and retinol To confirm the vitamin A production capacity of the bovine isolates inferred through UV spectroscopy, UHPLC-MS/MS analysis was performed (Figure 2). The ideal conditions (37°C, anaerobic and dark incubation on a rotary shaker at 150 rpm) optimized through several experiments were used. Though the extra carbon supplementation improves the b-carotene production, the UHPLC study was conducted without any supplementation to find out the original metabolic potential of the bovine bacterial strains. The calibration curve slopes and the coefficients of the standard solutions with b-carotene and retinol were calculated (Table 2).

Table 2. MS/MS MRM ions and linearity of b-carotene and retinol in UHPLC/MS- MS

Linearity with the best fit is established by least-squares regression analysis. The linear concentration range is from 0.05 to 100 mg/I for b-carotene and from 0.05 to 500 mg/I for retinol. The lowest detection limits are 0.01 mg/I and 0.03 mg/I for b- carotene and retinol with an injection volume of 5 mI_. The presence of b-carotene, retinol, and retinyl acetate was confirmed and quantified with 535.43 m/z, 269.22 m/z, and 269.23 m/z, respectively. Figure 2 indicates the UHPLC-MS/MS chromatogram peaks of the bacterial extract in the mobile phase. The retention times for retinol, retinyl acetate, and b-carotene are 4.23, 6.10, and 11.58, respectively. The retinyl acetate was added to the bacterial pellet before extraction to check the recovery of the vitamin A compounds in the extraction process and calculated accordingly (Schaffer et al. 2010).

Table 3. b-carotene production of probiotic bacteria from bovine and other sources ¹

¹ The b-carotene content of the bovine bacterial isolates reported in this study was measured through UHPLC-MS/MS. SD denotes standard deviation. The values with different superscript letters differ significantly (P<0.05). "This is the first study publishing the natural b-carotene producing E. coli.

The b-carotene content of the bacterial extracts from the bovine source is compared to the results of other sources and tabulated in Table 3. The strain DSM 16365T isolated from bovine liver produces 111 .95±3.10 pg b-carotene per g dry cell weight, whereas other strains are shown to produce 22.82 to 53.26 pg/g dry cell weight. The two-fold production difference among the bovine Lactobacillus strains may be due to strain-specific metabolic characteristics or distinct gene expression mechanisms. The strain-specific metabolic potential of bacterial carotenoid production may be due to the overexpression or interruptions in an operon with genes such as crtN and crtM where the vitamin biosynthetic enzymes are clustered together. Most lactic acid bacteria have the potential to produce more C40 carotenoids than C30 which might be due to the presence of genes coding for geranylgeranyl pyrophosphate (GGPP) and appropriate cyclase enzymes which might have acquired from their habitats. The current study data also corroborates the bacterial carotenoid production described in L. plantarum strain LTH4936. The concept of shared mechanisms in some well-studied bacteria like Lactobacillus with generally regarded as safe (GRAS) status supports this finding and the experiments of probiotic bacteria from animal sources like bovine liver. Though bacterial strains like Staphylococcus, Streptococcus, and Bacillus in rat gut are found to synthesize carotenoids, there is no study reporting E. coli naturally producing b- carotene. The bovine E. coli strain DSM 682 produces 44.77±2.08 pg/g of b-carotene in the anaerobic incubation. The retinol producing capacity of the five bovine isolates was evaluated with and without supplementation of 300 ppm b-carotene. The growth medium and conditions were maintained similar to b-carotene production. Some of the bovine isolates produced a small amount of retinol (Table 4).

Table 4. Retinol production from bovine bacteria with and without beta-carotene supplementation analysed through UHPLC-MS/MS analysis ^{1 1} The retinol content of the bovine bacterial isolates measured through UHPLC- MS/MS. SD denotes standard deviation. The values with different superscript letters differ significantly (P < 0.05).

DSM 16365T produces 210.74 ± 3.12 and 31.29 ± 0.95 ppm of retinol with and without the addition of 300 ppm of b-carotene in the growth medium through UHPLC-MS/MS analysis. Interestingly, the bovine E. coli DSM 682 also produces 55.17± 1 .98 ppm of retinol which is the very first natural retinoid producing strain from a cattle origin. The retinol production of the five strains was monitored through UV spectroscopy, and it was found that the maximum absorption is at 24 h, which begins to decline with more incubation time. The retinol deterioration in a bacterial culture medium may be due to cellular oxidative degradation and culture temperature. Susceptibility and adaptive responses to environmental stress, gastric juice, and bile salt

An ideal probiotic strain with commercial potential should resist environmental stress conditions during fermentation-like processes such as variable temperature, ethanol and salt concentrations. The tolerance of the five bovine isolates screened through UHPLC-MS/MS analysis with good vitamin A producing capacity was monitored (OD6OO>0.1 ) in different incubation temperature, and various concentrations of ethanol and NaCI after 24 h of anaerobic incubation. Figure 3 shows that all the five bovine strains grow well at a high temperature (45 °C) and at 37 °C, although not as good as that of 10 °C and 4 °C. The growth at a high temperature of 45 °C has been reported in few Lactobacillus strains during probiotic research, and that is good for brewing industries.

Concerning the capability to grow at a high concentration of ethanol, the strain isolated from the bovine liver DSM 16365T, and bovine intestine DSM 12028 exhibits better tolerance at 15% of ethanol (Figure 3) which is necessary for the dairy as well as wine industries. The bovine probiotic isolates are better at tolerating the high osmotic concentration of around 5% (Figure 3), and that is a prerequisite for various food product developments such as balancing the pH during fermentation. In a 0.3% bile tolerance assay, all the bovine bacteria exhibit a better tolerance even after four hours. However, the bovine E. coli DSM 682 shows significant differences in bile tolerance and other environmental stress conditions compared to Lactobacillus strains (P< 0.05). Overall, L. plantarum ssp. argentoratensis DSM 16365T shows better tolerance in all the environmental conditions.

The major physiological challenges in surviving the passage through the gastrointestinal tract and stomach are low pH and the antimicrobial action of pepsin and gastric juice. Here, the effect of simulated gastric juice different pH (2.0, 3.0, and 4.0) on the viability of the bovine isolates for 4 h shows no significant viability loss throughout the incubation period using multivariate ANOVA analysis (Table 5).

Table 5. Tolerance of the bovine isolates to simulated gastric juices (pH 2.0, 3.0, 4.0) ¹

¹ The viable count of bacteria in their selective media was converted to logio values. The interaction among the isolate, pH, and bacterial colony count at different times were calculated through multivariate ANOVA, and there was no statistically significant difference found at P<0.05.

E. coli DSM 682 isolated from the bovine intestine shows poor resistance but does not lose complete viability in the gastric juice environment. The potential of lactic acid bacteria varies based on strain and or source-specific characteristics. It has been reported that most of the probiotic Lactobacillus species exhibit good survival at pH 3.0. Thus, in this study bovine Lactobacillus isolates with high acid tolerance are likely to survive gastric conditions. The PCA bi-plot (Figure 4) describes the interrelations of the probiotic characteristics and vitamin A (b-carotene and retinol) production of the bovine isolates. The variables with factor loading values of more than 0.5 were selected for the bi-plot. The PCA bi plot shows that 86.53% of the total variation is explained by the two principal components (F1 = 58.75% and F2 = 27.78%). In terms of the probiotic characteristics and vitamin A compound production capacity, L. plantarum DSM 16365T shows a positive association with growth at 10 °C and is distinct from other bovine strains in the production of vitamin A compounds. However, the growth at 5% NaCI, 0.3% bile salt, 5 and 15% ethanol and 45 °C is clustered together and in the positive axis of both F1 and F2.

Conclusion

The present investigation of b-carotene and retinol producing bovine bacteria with GRAS status and an inhabitant of the human gastrointestinal tract has gained special interest due to its potential to develop probiotic and/or synbiotic food for vitamin A biofortification. There is enthusiasm for finding natural b-carotene producing gut- friendly bacteria to combat VADD. It is shown here that probiotic bacteria from animal origin can synthesize b-carotene and/or retinol, which could be developed into a synbiotic food, and that the probiotics will colonize with the human gut cells to synthesize the vitamin A.

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