D-/L-Lactic Acid (D-/L-Lactate) (Rapid) Assay Kit

Many of the enzymatic test kits are official methods of prestigious organisations such as the Association of Official Analytical Chemicals (AOAC) and the American Association of Cereal Chemists (AACC) in response to the interest from oenologists. Megazyme decided to use its long history of enzymatic bio-analysis to make a significant contribution to the wine industry, by the development of a range of advanced enzymatic test kits. This task has now been successfully completed through the strategic and comprehensive process of identifying limitations of existing enzymatic bio-analysis test kits where they occurred, and then using advanced techniques, such as molecular biology (photo 1), to rapidly overcome them. Novel test kits have also been developed for analytes of emerging interest to the oenologist, such as yeast available nitrogen (YAN; see pages 2-3 of issue 117 article), or where previously enzymes were simply either not available, or were too expensive to employ, such as for D-mannitol analysis.

It is without doubt that testing plays a pivotal role throughout the whole of the vinification process. To produce the best possible quality wine and to minimise process problems such as “stuck” fermentation or troublesome infections, it is now recognised that if possible testing should begin prior to harvesting of the grapes and continue through to bottling. Traditional methods of wine analysis are often expensive, time consuming, require either elaborate equipment or specialist expertise and frequently lack accuracy. However, enzymatic bio-analysis enables the accurate measurement of the vast majority of analytes of interest to the wine maker, using just one piece of apparatus, the spectrophotometer (see previous issue No. 116 for a detailed technical review). Grape juice and wine are amenable to enzymatic testing as being liquids they are homogenous, easy to manipulate, and can generally be analysed without any sample preparation.

Two Gram-stain-positive bacterial strains, designated 213-9(3)^T and 30-1(2)^T, were isolated from traditional Chinese pickle, and were characterized using a polyphasic taxonomic approach. Results of 16S rRNA gene sequence analysis indicated that strain 213-9(3)^T was most closely related to Levilactobacillus paucivorans TMW 1.1424^T, Levilactobacillus huananensis 151-2B^T and Levilactobacillus lindianensis 220-4^T, having 99.7–99.9 % 16S rRNA gene sequence similarities; strain 30-1(2)^T was most closely related to Lapidilactobacillus achengensis 247-4^T, with 99.4 % 16S rRNA gene sequence similarity. Strain 213-9(3)^T shared the highest pheS (93.9 %), rpoA (99.3 %) and concatenated pheS and rpoA (97.5 %) sequence similarities to L. paucivorans TMW 1.1424^T. Strain 30-1(2)^T had the highest pheS (82.4 %), rpoA (95.5 %) and concatenated pheS and rpoA (91.2 %) sequence similarities to L. achengensis 247-4^T. The phylogenetic relationships based on concatenated pheS and rpoA sequences and whole genome sequences were identical to those based on 16S rRNA gene sequences. Strain 213-9(3)^T exhibited the highest average nucleotide identity (ANI) and digital DNA–DNA hybridization (dDDH) values (92.7 and 48.8 %, respectively) to L. paucivorans DSM 22467^T. Strain 30-1(2)^T had the highest ANI (84.4 %) and dDDH (32.8 %) values with L. achengensis 247-4^T. Acid production from d-galactose, d-glucose, d-mannose, N-acetyl-β-glucosaminidase, arbutin, salicin, cellobiose, maltose, gentiobiose, d-tagatose and gluconate, hydrolysis of aesculin, and activity of cystine arylamidase could differentiate strain 213-9(3)^T from L. paucivorans DSM 22467^T. Acid production from l-arabinose, d-ribose, d-xylose and d-galactose, and activity of alkaline phosphatase, esterase (C4), α-mannosidase and α-fucosidase could differentiate strain 30-1(2)^T from L. achengensis 247-4^T. Based upon the data obtained in the present study, two novel species, Levilactobacillus humaensis sp. nov. and Lapidilactobacillus luobeiensis sp. nov., are proposed and the type strains are 213-9(3)^T (=CCM 9241^T=CCTCC AB 2022115^T=JCM 35554^T) and 30-1(2)^T (=CCM 9240^T=CCTCC AB 2022114^T=JCM 35553^T), respectively.

High optical purity lactic acid is in high demand as the precursor for synthesizing polylactic acid (PLA). The costs of expensive carbohydrates and nitrogen source materials accounts for a large portion of the production costs in lactic homo-fermentation. The use of lignocellulosic biomass for lactic acid production reduces the cost of the carbohydrate feedstock, but the cost of nitrogen sources is a big challenge when considering the high prices of general nitrogen sources. Low-cost nitrogen materials are vulnerable to being contaminated by exogenous mixed L-lactic acid and D-lactic acid; thus, their feasibility as nitrogen sources for the production of optically pure lactic acid products is hindered. The available reports focus on cost reduction using agro-industrial byproducts as nutrient sources, with these presenting fewer concerns on the effect of the optical purity of lactic acid-product monomers for polymerization. In this study, commonly used low-cost nutrient sources were characterized and screened for high optical purity L-lactic acid fermentation. Corn steep liquor (CSL), a widely used and cheap nutrient for lactic acid fermentation, was found not to be suitable because of its high content of mixed D-/L-lactic acids (up to 20%, w/w). On the other hand, cottonseed meal was found to be completely free of mixed L-/D-lactic acids. Therefore, the cottonseed meal was hydrolyzed with dilute sulfuric acid and used as a nitrogen source for L-lactic acid fermentation using lignocellulose feedstock as a substitution for yeast extract and peptone. The results showed that the final L-lactic acid titer reached 96.5 ± 0.2 g/L from 25% (w/w)-solids loaded pretreated and biodetoxified wheat straw with a yield of 0.31 g/g feedstock and an optical purity of 99.7%. The techno-economic evaluation indicated that the cost of the cottonseed meal was only USD 0.193/kg of lactic acid product, and the minimum lactic acid selling price (MLSP) was USD 0.813/kg of lactic acid product, which was only 25.1% compared to the use of yeast extract and peptone as the nutrients. Cellulosic L-lactic acid production using cottonseed meal as a complex nutrient source showed competitive performance when compared to starch feedstock from food crops.

New technologies development and renewable source exploitation are key tools to realize the European Green Deal and to boost the bio-based economy. In this context, fermentation of organic residues as food waste is an efficient method to obtain marketable products such as carboxylic acids widely applied in industrial production. Under favourable thermodynamic conditions, short chain fatty acids deriving from primary fermentation could be biologically converted into medium-chain fatty acids as caproate via chain elongation (CE) process, by using ethanol or lactate as electron donors. This study evaluates the effectivity of producing caproate from Food Waste extract rich in organics with in situ electron donor production. The test carried out at OLR 15 gCOD L⁻¹d⁻¹ showed high Volatile Fatty Acids (from acetic to caproic acid) yields (0.37 g g⁻¹COD_fed), with a maximum caproate concentration of 8 g L⁻¹. The associated microbiome was composed by lactate-producing bacteria (Corynebacterium, Lactobacillus, and Olsenella) and by chain elongators (Clostridiaceae and Caproiciproducens). By stressing the system with OLR increase up to 20 gCOD L⁻¹d⁻¹, the CE process was inhibited by the high concentration of caproate (low occurrence of Clostridiaceae and Caproiciproducens). Nevertheless, after few days of stop-feeding regime imposed to the system, the microbiome restored its capability to proceed with lactate-based CE pathways. Different batch tests carried out with the inhibited biomass at increasing initial caproate concentration confirmed its impact on lactate utilization kinetics.

Lactic acid is an essential organic chemical in food, clinical, chemical, and bioprocessing industries. We are reporting for the first time, a rapid and facile enzyme-free colorimetric sensor for the detection of lactic acid (LA) in food samples using nitrophenol (p-NP) added copper nanoparticles (CuNPs). The responses of p-NP (5 mmol/L) to lactic acid in the presence of CuNPs were reported by analysing the UV-VIS absorption spectra in an aqueous solution. CuNPs were synthesized by the chemical reduction method and were identified to be in the Cubic phase using XRD analysis. The sample characterization studies were performed using the SEM, UV for obtaining good optical and selective lactic acid-sensing properties. The sensing mechanism is due to the aggregation responses of CuNPs conjugated p-NP in the presence of lactic acid as seen in the UV-VIS absorption spectra shift studied in an aqueous solution. The absorption spectrum of p-NP exhibited one intense band at 402 nm in the presence of CuNPs. Although the addition of lactic acid may cause π –π* transitions led to a hypsochromic shift (blue shift) with a subtle decrease in the wavelength from 402 to 315 nm, the solution underwent from being colored (greenish-yellow) to colorless. The stoichiometric ratio of a binding event is explained using Job's plot. The naked eye colorimetric response of the sensor-enabled detection limit of lactic acid to 0.33 mM. The sensor studies were compared with standard LC-MS/MS revealing a good recovery (95%) in food samples. This method is facile and alternate to the current high-cost techniques and enzymatic sensors.

The aim of the present study was to investigate the feasibly of using traditional milk kefir grains for the production of water kefir-like beverages and assess the changes in the physicochemical characteristics and the microbial populations of the fermented beverages. To this end, experiments of milk fermentation were primarily conducted at different temperatures and upon selection of the optimal, a gradual substitution of the substrate was performed by replacing milk from a sucrose-based solution. After the successful fermentation of the sucrose substrate, fruit juices were used as fermentation substrates. Sensory evaluation of the sugar-based beverages was also performed in order to access their acceptability for consumption. According to the results, the transition from milk to water kefir is indeed feasible, leading to the production of beverages with relatively higher ethanol concentrations (up to 2.14 ± 0.12% w/v) than milk kefir and much lower lactic acid concentrations (up to 0.16 ± 0.01% w/v). During the fermentation of the sugary substrates, yeasts seemed to be dominant over lactic acid bacteria, in contrast to what was observed in the case of milk kefir, where LAB dominated. The sensory evaluation revealed that all sugar-based beverages were acceptable for consumption, with the fruit-based ones obtaining, though, a better score in all attributes.

A photonic crystal fiber (PCF)-based fluorescence sensor is developed for rapid and sensitive detection of lactic acid (LA) enantiomers in serum samples. The sensor is fabricated by chemical binding dual enzymes on the inner surface of the PCF with numerous pore structures and a large specific surface area, which is suitable to be utilized as an enzymatic reaction carrier. To achieve simultaneous detection of L-LA and D-LA, the PCF with an aldehyde-activated surface is cut into two separate pieces, one of which is coated with L-LDH/GPT enzymes and the other with D-LDH/GPT enzymes. By being connected and carefully aligned to each other by a suitable sleeve tube connector, the responses of both L-LA and D-LA sensors are determined by laser-induced flourescence (LIF) detection. With the aid of enzyme-linked catalytic reactions, the proposed PCF sensor can greatly improve the sensitivity and analysis speed for the detection of LA enantiomers. The PCF sensor exhibits a low limit of detection of 9.5 μM and 0.8 μM, and a wide linear range of 25-2000 μM and 2-400 μM for L-LA and D-LA, respectively. The sensor has been successfully applied to accurate determination of LA enantiomers in human serum with satisfactory reproducibility and stability. It is indicated that the present PCF sensors would be used as an attractive analytical platform for quantitative detection of trace-amount LA enantiomers in real biological samples, and thus would play a role in disease diagnosis and clinical monitoring in point-of-care testing.

L-lactate is an essential organic chemical in food processing, clinical, chemical, and fermentation industries. Titanium oxide nanoparticles (TiO₂ NPs) was synthesized by the green chemical method and structural, morphological, zeta potential characterization studies were performed for achieving lower limit of detection (LOD), stability, and sensor fabrication. First time reporting TiO₂ NPs functionalized with 3-aminophenylboronic acid (3-APBA^TiO₂ NPs) for L-lactate detection in apple puree (lab prepared) and commercial Tropicana (Indian local brand) apple juice samples. Nanocomposite in the aqueous phase (pH 7.4) shows a selective binding towards L-lactate that results in a blue shift of UV-VIS absorption wavelength from 300 nm to 288 nm as L-lactate binds to the boron atom of boronic acid and forms a tetragonal complex stabilized by the TiO₂ NPs. The apple samples spiked with L-Lactate (1-10 mM) showed an absorption peak intensity increases as per lactate concentration with the LOD= 3.11 mM using the calibration graphs. The nanosensor data was validated with a commercial Megazyme kit and conventional High-performance liquid chromatography (HPLC) technique. L-lactate content in commercial Tropicana apple juice samples was detected as 6.1 mM with 93.5 % correlation. This method could be an alternative to conventional L-lactate detection techniques and can be interfaced with a smartphone for onsite detection device development.

Changes in the nutritive value of forages are imminent under different ensiling conditions. Thus, a study was conducted to assess the effects of ensiling length and storage temperature on the nutritive value, fermentation characteristics and fibre-bound protein of three tropical forage legumes, sorghum and mixtures of sorghum and the legumes. Soybean (Glycine max), jack bean (Cannavalia ensiformis), lablab (Lablab purpureus) and sorghum (Sorghum bicolor) were solely grown and harvested, and the legumes were wilted before ensiling. Mixtures of sorghum and each legume were handmade on a percentage fresh weight basis of 60:40. Each forage and mixtures (400 g) were ensiled in polythene vacuum bags with homofermentative lactic acid bacteria inoculation for 30, 75 and 180 days. A set of mini silos were stored indoors, and another batch was stored outdoors. HOBO Pro v2 data loggers were deployed to monitor the ambient temperature of the storage locations during the entire ensiling period (from day 0-180). Measurements included nutrient analysis, fermentation quality and fibre bound protein characteristics. The hourly ambient temperature for outdoor and indoor storage ranged from 16° to 61°C vs 18-35°C, respectively. Proximate constituents of all silages were influenced by ensiling length. Significant changes were primarily detected in fermentation products of legume silages between 30 and 75 d of ensiling. There were reduced fermentation products for silages stored outdoors. The ensiling length influenced proportions of neutral detergent insoluble nitrogen (NDIN) and acid detergent insoluble nitrogen (ADIN) with outdoor silages resulting in a higher proportion of NDIN and ADIN compared to indoor silages. Overall, a short period of ensiling preserves the nutritional quality of ensiled forages compared to prolonged storage at high ambient temperatures typical of the tropics that increase nutrient losses. Thus, changes in the nutritional composition of forages during ensiling should be considered during ration formulations.

To increase the dry matter and metabolisable energy intake of cows, dairy farmers often supplement pasture with concentrates and conserved fodder. Feeding large amounts of highly fermentable concentrates to cows can result in metabolic issues, such as ruminal acidosis, and thus safer but more efficient introduction strategies are desirable. We assessed the role that forages play in ruminal, behavioural and production responses to a wheat grain challenge in dairy cows with no previous wheat adaptation. Multiparous lactating Holstein dairy cows (n = 16) were fed a forage-only diet of either lucerne (Medicago sativa) hay, perennial ryegrass (Lolium perenne L.) hay or one of two cultivars of zero-grazing fresh perennial ryegrass herbage (Bealey or Base), for 3 weeks. The forage diet was then supplemented with crushed wheat grain at 8 kg dry matter/cow day⁻¹, with no adaptation period. Wheat comprised between 32 and 43% of total dry matter intake. Cows fed hay maintained a higher mean ruminal fluid pH than those fed herbage, on both the forage-only diet (6.43 vs. 6.17) and the forage plus wheat diet (6.03 vs. 5.58). Following supplementation of wheat, cows fed herbage exhibited minimum ruminal fluid pH levels indicative of acute ruminal acidosis, at 5.15 and 5.06 for cultivars Bealey and Base, respectively. Furthermore, for both herbage cultivars, adding wheat resulted in a ruminal fluid pH under 6 for >20 h/day. The ruminal environment of cows fed lucerne hay remained most stable throughout the grain challenge, spending the least amount of time below pH 6.0 (9.0 h/day). Hay created a ruminal environment that was better able to cope with the accumulation of acid as wheat was digested. A combination of increased ruminating time and a slower rate of fermentation, due to higher neutral detergent fiber and lower metabolisable energy concentrations in the hays, is likely responsible for the higher ruminal fluid pH values. Forage plays a critical role in wheat introduction strategies; aggressive adaptation strategies could be implemented when a hay such as lucerne is used as the base forage.

With the aim to reach the maximum recovery of bulk and specialty bioproducts while minimizing waste generation, a multi-product biorefinery for ethanol and lactic acid production from the biomass of cyanobacterium Arthrospira platensis was investigated. Therefore, the residual biomass resulting from different pretreatments consisting of supercritical fluid extraction (SF) and microwave assisted extraction with non-polar (MN) and polar solvents (MP), previously applied on A. platensis to extract bioactive metabolites, was further valorized. In particular, it was used as a substrate for fermentation with Saccharomyces cerevisiae LPB-287 and Lactobacillus acidophilus ATCC 43121 to produce bioethanol (BE) and lactic acid (LA), respectively. The maximum concentrations achieved were 3.02 ± 0.07 g/L of BE by the MN process at 120 rpm 30°C, and 9.67 ± 0.05 g/L of LA by the SF process at 120 rpm 37°C. An economic analysis of BE and LA production was carried out to elucidate the impact of fermentation scale, fermenter costs, production titer, fermentation time and cyanobacterial biomass production cost. The results indicated that the critical variables are fermenter scale, equipment cost, and product titer; time process was analyzed but was not critical. As scale increased, costs tended to stabilize, but also more product was generated, which causes production costs per unit of product to sharply decrease. The median value of production cost was US$ 1.27 and US$ 0.39, for BE and LA, respectively, supporting the concept of cyanobacterium biomass being used for fermentation and subsequent extraction to obtain ethanol and lactic acid as end products from A. platensis.

Enterococcus spp., known for their wide ecological distribution, have been associated with various fermented food products of plant and animal origin. The strains used in this study, bacteriocinogenic Enterococcus faecium previously isolated from artisanal soybean paste, have shown strong activity against Listeria spp. and vancomycin-resistant enterococci. Although their antimicrobial activity is considered beneficial, the potential application of enterococci is still under debate due to concerns about their safety for human and other animal consumption. Therefore, this study not only focuses on the screening of potential virulence factors, but also the auxiliary beneficial properties of the strains Ent. faecium ST651ea, ST7119ea, and ST7319ea. Phenotypic screening for gelatinase, hemolysin, and biogenic amine production showed that the strains were all safe. Furthermore, the antibiogram profiling showed that all the strains were susceptible to the panel of antibiotics used in the assessment except for erythromycin. Yet, Ent. faecium ST7319ea was found to carry some of the virulence genes used in the molecular screening for safety including hyl, esp, and IS16. The probiotic potential and other beneficial properties of the strains were also studied, demonstrating high aggregation and co-aggregation levels compared to previously characterized strains, in addition to high survivability under simulated gastrointestinal conditions, and production of numerous desirable enzymes as evaluated by APIZym, indicating diverse possible biotechnological applications of these strains. Additionally, the strains were found to carry genes coding for γ-aminobutyric acid (GABA) production, an auxiliary characteristic for their probiotic potential. Although these tests showed relatively favorable characteristics, it should be considered that these assays were carried out in vitro and should therefore also be assessed under in vivo conditions.

We aimed to assess the effect of feeding Bacillus subtilis C-3102 on the growth and rumen microbiota in the preweaned calves. Twelve newborn Japanese Black calves were randomly allocated to either the control (n = 6) or the treatment (n = 6) groups in the present study. Calves in the treatment group were offered B. subtilis C-3102 supplemented milk replacer throughout the preweaning period. Rumen fermentation during the first 21 days of life seemed to be slightly suppressed by feeding B. subtilis C-3102. This fermentation shift was probably attributed to the lower abundance of the core members of rumen microbiota until 21 days of age in the calves fed B. subtilis C-3102. However, feeding B. subtilis C-3102 did not influence the abundance of the core members of rumen microbiota at 90 days of age. Distribution of Sharpea spp. and Megasphaera spp., which potentially contribute to low methane production and are regarded as beneficial rumen bacteria, was higher in the rumen of calves fed B. subtilis C-3102 at 90 days of age. These results suggest that B. subtilis C-3102 supplementation in milk replacer could potentially contribute to the improvement of feed efficiency after weaning via the establishment of beneficial rumen bacteria.

A comprehensive study into the potential of bioprocessing techniques (sprouting and sourdough fermentation) for improving the technological and nutritional properties of wheat breads produced using barley and lentil grains was undertaken. Dextran biosynthesis in situ during fermentation of native or sprouted barley flour (B or SB) alone or by mixing SB flour with native or sprouted lentil flour (SB-L or SB-SL) by Weissella paramesenteroides SLA5, Weissella confusa SLA4, Leuconostoc pseudomesenteroides DSM 20193 or Weissella confusa DSM 20194 was assessed. The acidification and the viscosity increase during 24 h of fermentation with and without 16% sucrose (on flour weight), to promote the dextran synthesis, were followed. After the selection of the fermentation parameters, the bioprocessing was carried out by using Leuconostoc pseudomesenteroides DSM 20193 (the best LAB dextran producer, up to 2.7% of flour weight) and a mixture of SB-SL (30:70% w/w) grains, enabling also the decrease in the raffinose family oligosaccharides. Then, the SB-SL sourdoughs containing dextran or control were mixed with the wheat flour (30% of the final dough) and leavened with baker’s yeast before baking. The use of dextran-containing sourdough allowed the production of bread with structural improvements, compared to the control sourdough bread. Compared to a baker’s yeast bread, it also markedly reduced the predicted glycemic index, increased the soluble (1.26% of dry matter) and total fibers (3.76% of dry matter) content, giving peculiar and appreciable sensory attributes.

In our previous studies, we revealed the effect of lactose inclusion in calf starters on the growth performance and gut development of calves. We conducted the present study as a follow-up study to identify the shift in rumen microbiota and its relation to rumen fermentation when calves are fed a lactose-containing starter. Thirty Holstein bull calves were divided into 2 calf starter treatment groups: texturized calf starter (i.e., control; n = 15) or calf starter in which starch was replaced with lactose at 10% (i.e., LAC10; n = 15) on a dry matter basis. All calves were fed their respective treatment calf starter ad libitum from d 7, and kleingrass hay from d 35. Rumen digesta were collected on d 80 (i.e., 3 wk after weaning) and used to analyze rumen microbiota and fermentation products. There was no apparent effect of lactose feeding on the α-diversity and overall composition of rumen microbiota. Amplicon sequencing and real-time PCR quantification of the 16S rRNA gene confirmed that the abundance of butyrate-producing bacteria (i.e., Butyrivibrio group and Megasphaera elsdenii) did not differ between the control and LAC10 groups. Conversely, the relative abundance of Mitsuokella spp., which produce lactate, succinate, and acetate, was significantly higher in the rumen of calves that were fed lactose, whereas the lactate concentration did not differ between the control and LAC10 groups. These findings suggest that the lactate production can be elevated by an increase of Mitsuokella spp. and then converted into butyrate, not propionate, since the proportion of propionate was lower in lactose-fed calves. In addition, we observed a higher abundance of Coriobacteriaceae and Pseudoramibacter-Eubacterium in the LAC10 group. Both these bacterial taxa include acetate-producing bacteria, and a positive correlation between the acetate-to-propionate ratio and the abundance of Pseudoramibacter-Eubacterium was observed. Therefore, the higher abundance of Coriobacteriaceae, Mitsuokella spp., and Pseudoramibacter-Eubacterium in the rumen of lactose-fed calves partially explains the increase in the proportion of rumen acetate that was observed in our previous study.

Spent sulfite liquor (SSL), a waste stream from wood pulp production, has great potential as carbon source for future industrial fermentations. In the present study, SSL was separated into a hemicellulose derived sugar syrup (HDSS) and a lignosulfonic fraction by simulated moving bed chromatography. The recovery of SSL sugars in the HDSS was 89% and the fermentation inhibitors furfural, 5-hydroxymethylfurfural and acetic acid were removed by 98.7%, 60.5% and 75.5%, respectively. The obtained sugars have been converted to L-lactic acid, a building block for bioplastics, by fermentation with the lactic acid bacterium Enterococcus mundtii DSM 4838. Batch fermentations on HDSS produced up to 56.3 g/L L-lactic acid. Simultaneous conversion of pentose and hexose sugars during fed-batch fermentation of wildtype E. mundtii led to 87.9 g/L optically pure (>99%) L-lactic acid, with maximum productivities of 3.25 g/L.h and yields approaching 1.00 g/g during feeding phase from HDSS as carbon source.

A mechanistic, spatio-temporal model to predict early stage semi-solid food ripening, exemplary for semi-solid casein matrices, was created using software based on the finite element method (FEM). The model was refined and validated by experimental data obtained during 8 wk of ripening of a casein matrix that was inoculated by one single central injection of starter culture. The resulting spatio-temporal distributions of lactococci strains, lactose, lactic acid/lactate and pH allowed us to optimize a number of parameters of the predictive model. Using the optimized model, the agreement between simulation and experiment was found to be satisfactory, with the pH matching best. The predictive model unveiled that effective diffusion of substrate and metabolites were crucial for an eventual homogeneous distribution of the measured substances. Hence, while using the optimized parameters from the single injection model, an injection technology for starter culture to inoculate and ferment casein matrices homogeneously was developed by means of solving another optimization problem with respect to injection positions. The casein matrix inoculated by the proposed injection pattern (21 injections, distance = 19 mm) showed sufficient homogeneity (bacterial activity and pH distribution) after the early stages of ripening, demonstrating the potential of application of the injection technology for fermentation of casein-based foods e.g. cheese.

Three strains (YZ01^T, YZ02 and YZ03) of Gram-stain-positive, facultatively anaerobic rods were isolated from the forestomach contents collected from a captive male proboscis monkey (Nasalis larvatus) at Yokohama Zoo in Japan. Phylogenetic analysis of the 16S rRNA gene sequences revealed that these strains belonged to the genus Lactobacillus. Based on the sequence similarity of the 16S rRNA gene, Lactobacillus delbrueckii subsp. indicus JCM 15610^T was the closest phylogenetic neighbour to YZ01^T. Sequence analyses of two partial concatenated housekeeping genes, the RNA polymerase alpha subunit (rpoA) and phenylalanyl-tRNA synthase alpha subunit (pheS) also indicated that the novel strains belonged to the genus Lactobacillus. The average nucleotide identity and digital DNA-DNA hybridization (dDDH) between L. delbrueckii subsp. indicus and YZ01^T were 85.9 and 31.4 %, respectively. The phylogenetic tree based on the whole genomic data of strains YZ01^T, YZ02 and YZ03 suggested that these three strains formed a single monophyletic cluster in the genus Lactobacillus, indicating that it belonged to a new species. The DNA G+C content of strain YZ01^T was 51.6 mol%. The major fatty acids were C_{16 : 0} and C_{18 : 1} ω9c. Therefore, based on phylogenetic, phenotypic and physiological evidence, strains YZ01^T, YZ02 and YZ03 represent a novel species of the genus Lactobacillus, for which the name Lactobacillus nasalidis sp. nov. is proposed with the type strain YZ01^T (=JCM 33769^T=DSM 110539^T).

A rod-shaped, facultative anaerobic, Gram-stain-positive bacteria, isolated from the cecum of a mini-pig, was designated as strain YH-lac23^T. Analysis of 16S rRNA gene sequences revealed that the strain was closely related to Lacticaseibacillus daqingensis JCM 33273^T (97.9 %), Lacticaseibacillus porcinae KCTC 21027^T (96.2 %) and Lacticaseibacillus manihotivorans KCTC 21010^T (95.7 %). Analysis of housekeeping gene sequences (pheS and recA) revealed that the strain formed a sub-cluster with L. daqingensis. The average nucleotide identity value for YH-lac23^T and its most closely related strain (L. daqingensis) is 80.7 %. The main fatty acids are C_{18 : 1}ω9c and C_{16 : 0}. The cell wall contains the peptidoglycan of meso-diaminopimelic acid. The G+C content of the genomic DNA is 59.8 mol%. In view of the chemotaxonomic, phenotypic and phylogenetic properties, YH-lac23^T (=KCTC 25006=JCM 33998) represents a novel taxon. The name Lacticaseibacillus absianus sp. nov. is proposed.