Combining selected immunomodulatory Propionibacterium freudenreichii and Lactobacillus delbrueckii strains: Reverse engineering development of an anti‐inflammatory cheese

Scope : Inflammatory bowel disease (IBD) constitutes a growing public health concern in western countries. Bacteria with anti‐inflammatory properties are lacking in the dysbiosis accompanying IBD. Selected strains of probiotic bacteria with anti‐inflammatory properties accordingly alleviate symptoms and enhance treatment of ulcerative colitis in clinical trials. Such properties are also found in selected strains of dairy starters such as Propionibacterium freudenreichii and Lactobacillus delbrueckii (Ld). We thus investigated the possibility to develop a fermented dairy product, combining both starter and probiotic abilities of both lactic acid and propionic acid bacteria, designed to extend remissions in IBD patients. Methods and results : We developed a single‐strain Ld‐fermented milk and a two‐strain P. freudenreichii and Ld‐fermented experimental pressed cheese using strains previously selected for their anti‐inflammatory properties. Consumption of these experimental fermented dairy products protected mice against trinitrobenzenesulfonic acid induced colitis, alleviating severity of symptoms, modulating local and systemic inflammation, as well as colonic oxidative stress and epithelial cell damages. As a control, the corresponding sterile dairy matrix failed to afford such protection. Conclusion : This work reveals the probiotic potential of this bacterial mixture, in the context of fermented dairy products. It opens new perspectives for the reverse engineering development of anti‐inflammatory fermented foods designed for target populations with IBD, and has provided evidences leading to an ongoing pilot clinical study in ulcerative colitis patients.     

Multilocus sequence typing of Propionibacterium freudenreichii

Propionibacterium freudenreichii is used as a ripening culture in Swiss cheese manufacture. This study investigates the molecular diversity and the population structure of this bacterium via multilocus sequence typing (MLST). Internal fragments of seven genes sequenced for 113 strains of different subspecies and origins allowed the resolution of 46 sequence types (STs) with occurrence frequencies ranging from 1 to 11. The core genome of the species harbours a low level of nucleotide polymorphism. In our data, single nucleotide polymorphisms account for only 2.28% of the concatenated sequences, and the average polymorphism rate in pairwise comparisons is 0.46%. The analyses reveal quantitatively comparable contributions of recombination and mutation in nucleotide changes at core genome loci along cell lineages. Remarkably, the STs exhibit little if any dairy biotope specialization. Phenotypic characterisation of the strains, based on their aptitude to use lactose and nitrate, shows that the two previously identified subspecies (freudenreichii and shermani) do not reflect the ancestral relationships in the P. freudenreichii population. The considerable phenotypic heterogeneity, found even at the ST level, suggests instead a history of recurrent switches between phenotypes.     

The growth of Propionibacterium cyclohexanicum in fruit juices and its survival following elevated temperature treatments

This study investigated the growth of Propionibacterium cyclohexanicum in orange juice over a temperature range from 4 to 40 degrees C and its ability to multiply in tomato, grapefruit, apple, pineapple and cranberry juices at 30 and 35 degrees C. Survival after 10 min exposure to 50, 60, 70, 80, 85, 90 and 95 degrees C in culture medium and in orange juice was also assessed. In orange juice the organism was able to multiply by 2 logs at temperatures from 4 to 35 degrees C and survived for up to 52 days. However, at 40 degrees C viable counts were reduced after 6 days and no viable cells isolated after 17 days. The optimum growth temperature in orange juice over 6 days was 25 degrees C but over 4 days it was 35 degrees C. The growth of P. cyclohexanicum was monitored in tomato, grapefruit, cranberry, pineapple and apple juices at 30 and 35 degrees C over 29 days. Cranberry, grapefruit and apple juice did not support the growth of P. cyclohexanicum. At 30 degrees C no viable cells were detected after 8 days in cranberry juice or after 22 days in grapefruit juice while at 35 degrees C no viable cells were detected after 5 and 15 days, respectively. However, in apple juice, although a 5 log reduction occurred, viable cells could be detected after 29 days. P. cyclohexanicum was able to multiply in both tomato and pineapple juices. In tomato juice, there was a 2 log increase in viable counts after 8 days at 30 degrees C but no increase at 35 degrees C, while in pineapple juice there was a 1 log increase in numbers over 29 days with no significant difference between numbers of viable cells present at 30 and 35 degrees C. The organism survived at 50 degrees C for 10 min in culture medium without a significant loss of viability while similar treatment at 60, 70 and 80 degrees C resulted in approximately a 3-4 log reduction, with no viable cells detected after treatment at 85 or 90 or 95 degrees C but, when pre-treated at intermediate temperatures before exposure to higher temperatures, some cells survived. However, in orange juice a proportion of cells survived at 95 degrees C for 10 min without pre-treatment and there was no significant difference between numbers surviving with and without pre-treatment. The results from this study demonstrate that P. cyclohexanicum is able to grow in a number of juices, other than orange juice, and able to survive a number of high temperature procedures. Therefore, if initially present in the raw materials P. cyclohexanicum might survive the pasteurization procedures used in the fruit juice industry, contaminate and consequently spoil the final product.     

Progress in genomics, metabolism and biotechnology of bifidobacteria

Members of the genus Bifidobacterium were first described over a century ago and were quickly associated with a healthy intestinal tract due to their numerical dominance in breast-fed babies as compared to bottle-fed infants. Health benefits elicited by bifidobacteria to its host, as supported by clinical trials, have led to their wide application as probiotic components of health-promoting foods, especially in fermented dairy products. However, the relative paucity of genetic tools available for bifidobacteria has impeded development of a comprehensive molecular understanding of this genus. In this review we present a summary of current knowledge on bifidobacterial metabolism, classification, physiology and genetics and outline the currently available methods for genetically accessing and manipulating the genus.     

一株费氏丙酸杆菌生长特性及其代谢物抑菌作用分析

对一株费氏丙酸杆菌生长特性及其代谢物抑菌作用进行研究。结果表明：该菌株生长周期约为10 d,第1～3天为其对数生长期,第4～8天为菌体生长的稳定期,第9～10天为衰亡期。在生长过程中,菌体利用糖的速率很快,还原糖在发酵1 d内几乎被消耗殆尽,发酵液pH值在发酵的第1天下降到4.6左右,此后一直到第10天,pH值基本稳定在4.6左右。在同等条件下,该丙酸杆菌代谢物对大肠杆菌的抑制作用优于对金黄色葡萄球菌和酵母菌的抑制作用。对代谢产物的分析结果表明,发酵产物中抑菌物质除了丙酸之外,还存在其他物质,初步判定为细菌素。     

费氏丙酸杆菌费氏亚种在不同基质中转化生成共轭亚油酸的研究

研究了在MRS、乳酸钠和脱脂乳三种培养基质下温度、时间、底物浓等因素对P.freudenreichiissp.freudenreichii生成CLA的影响,结果表明P.freudenreichiissp.freudenreichii在MRS、乳酸钠和脱脂乳中均具有生成CLA的能力,三种培养基质中一定浓度的葵花籽油对P.freudenreichiissp.freudenreichii的生长都有明显的抑制作用,在MRS培养基中葵花籽油浓度为12mg/ml,菌液量10%(V/V),30℃培养24hCLA生成量最大为24.813μg/ml。     

Relationship between gastrointestinal tolerance and exopolysaccharide production of propionibacteria strains under different pH and bile conditions

In this study, we report the effects of exposure to simulated gastric and intestinal juices on the survival of six strains of dairy propionibacteria. Gastrointestinal tolerance of propionibacteria strains was highly variable, depending on the strain and pH (P < 0.05). Exopolysaccharides (EPS) production of six propionibacteria strains under different pH conditions and bile concentrations was also studied. A positive correlation was obtained between the EPS production quantity of the strains and tolerance to simulated gastric juices at pH 2.0, 3.0 (P < 0.05). This investigation showed that high EPS production may be important in the selection of probiotic strains for resistance to upper gastrointestinal system conditions.     

Standardization of milk using cold ultrafiltration retentates for the manufacture of Swiss cheese: effect of altering coagulation conditions on yield and cheese quality

Fortification of cheesemilk with membrane retentates is often practiced by cheesemakers to increase yield. However, the higher casein (CN) content can alter coagulation characteristics, which may affect cheese yield and quality. The objective of this study was to evaluate the effect of using ultrafiltration (UF) retentates that were processed at low temperatures on the properties of Swiss cheese. Because of the faster clotting observed with fortified milks, we also investigated the effects of altering the coagulation conditions by reducing the renneting temperature (from 32.2 to 28.3°C) and allowing a longer renneting time before cutting (i.e., giving an extra 5 min). Milks with elevated total solids (TS; ∼13.4%) were made by blending whole milk retentates (26.5% TS, 7.7% CN, 11.5% fat) obtained by cold (<7°C) UF with part skim milk (11.4% TS, 2.5% CN, 2.6% fat) to obtain milk with CN:fat ratio of approximately 0.87. Control cheeses were made from part-skim milk (11.5% TS, 2.5% CN, 2.8% fat). Three types of UF fortified cheeses were manufactured by altering the renneting temperature and renneting time: high renneting temperature = 32.2°C (UFHT), low renneting temperature = 28.3°C (UFLT), and a low renneting temperature (28.3°C) plus longer cutting time (+5 min compared to UFLT; UFLTL). Cutting times, as selected by a Wisconsin licensed cheesemaker, were approximately 21, 31, 35, and 32 min for UFHT, UFLT, UFLTL, and control milks, respectively. Storage moduli of gels at cutting were lower for the UFHT and UFLT samples compared with UFLTL or control. Yield stress values of gels from the UF-fortified milks were higher than those of control milks, and decreasing the renneting temperature reduced the yield stress values. Increasing the cutting time for the gels made from the UF-fortified milks resulted in an increase in yield stress values. Yield strain values were significantly lower in gels made from control or UFLTL milks compared with gels made from UFHT or UFLT milks. Cheese composition did not differ except for fat content, which was lower in the control compared with the UF-fortified cheeses. No residual lactose or galactose remained in the cheeses after 2 mo of ripening. Fat recoveries were similar in control, UFHT, and UFLTL but lower in UFLT cheeses. Significantly higher N recoveries were obtained in the UF-fortified cheeses compared with control cheese. Because of higher fat and CN contents, cheese yield was significantly higher in UF-fortified cheeses (∼11.0 to 11.2%) compared with control cheese (∼8.5%). A significant reduction was observed in volume of whey produced from cheese made from UF-fortified milk and in these wheys, the protein was a higher proportion of the solids. During ripening, the pH values and 12% trichloroacetic acid-soluble N levels were similar for all cheeses. No differences were observed in the sensory properties of the cheeses. The use of UF retentates improved cheese yield with no significant effect on ripening or sensory quality. The faster coagulation and gel firming can be decreased by altering the renneting conditions.     

Isolation and characterization of proline iminopeptidase from Propionibacterium freudenreichii ATCC 9614

A dimeric, 90 kDa subunit intracellular proline iminopeptidase from Propionibacterium freudenreichii ATCC 9614 was purified to homogeneity by chromatography on hydroxyapatite, Sephacryl 200, Phenyl Superose and Mono Q. The enzyme was specific on Pro-p-nitroanilide and Pro-X dipeptides. It hydrolyzed 2 fragments of hormone oligopeptides with an N-terminal proline: bradykinin, f2-7 and substance P, f4-11. A number of oligopeptides containing 5-11 amino acids residues and proline at the penultimate position from N-terminus or other internal position were not hydrolyzed. The enzyme was most active at pH 7-7.5 and at 37-40 degrees C but it retained 9% of maximal activity at pH 5.5 and >12% of maximal activity at 10 or 60 degrees C. The enzyme was inhibited strongly by the serine protease inhibitor 3,4-dichloroisocoumarin, and stimulated markedly by 1 mol/l of NaCl. The results indicate that the enzyme may lead to the accumulation of proline from dipeptides and oligopeptides during the ripening of cheese.     

费氏丙酸杆菌生物学特性及与5株益生菌拮抗性试验研究

为了评价费氏丙酸杆菌的益生特性,费氏丙酸杆菌活化后,划线和镜检观察其形态特征,然后利用体外共培养法测定费氏丙酸杆菌对3种致病菌的抑制效果,同时利用平板计数法研究费氏丙酸杆菌的耐酸性、耐胆盐和耐高温等生物学特性,以及5株益生菌拮抗性试验。结果表明,费氏丙酸杆菌对大肠杆菌和沙门氏菌没有显著的抑制作用,而对金黄色葡萄球菌的抑制效果明显（P＜0.01）;其在pH值为2.0条件下维持3 h,活菌数降低一个数量级;在胆盐含量为0.30%条件下维持6 h,存活率为18.67%;在60 ℃下处理20 min,存活率为12%,因此费氏丙酸杆菌具有一定的耐酸、耐高温能力以及很强的耐胆盐活性。除枯草芽孢杆菌与丁酸梭菌有拮抗作用外,其余菌株间均没有拮抗作用。     

Preliminary study of ultrasonic structural quality control of Swiss-type cheese

There is demand for a new nondestructive cheese-structure analysis method for Swiss-type cheese. Such a method would provide the cheese-making industry the means to enhance process control and quality assurance. This paper presents a feasibility study on ultrasonic monitoring of the structural quality of Swiss cheese by using a single-transducer 2 MHz longitudinal mode pulse-echo setup. A volumetric ultrasonic image of a cheese sample featuring gas holes (cheese-eyes) and defects (cracks) in the scan area is presented. The image is compared with an optical reference image constructed from dissection images of the same sample. The results show that the ultrasonic method is capable of monitoring the gas-solid structure of the cheese during the ripening process. Moreover, the method can be used to detect and to characterize cheese-eyes and cracks in ripened cheese. Industrial application demands were taken into account when conducting the measurements.     

Capsular exopolysaccharide biosynthesis gene of Propionibacterium freudenreichii subsp. shermanii

In the dairy industry, exopolysaccharides (EPS) contribute to improving the texture and viscosity of cheese and yoghurt and also receive increasing attention because of their beneficial properties for health. For lactic acid bacteria, the production of EPS is well studied. However, for dairy propionibacteria the biosynthesis of EPS is poorly documented. A polysaccharide synthase-encoding gene was identified in the genome of Propionibacterium freudenreichii subsp. shermanii TL 34 (CIP 103027). This gene best aligns with Tts, the polysaccharide synthase gene of Streptococcus pneumoniae type 37 that is responsible for the production of a beta-glucan capsular polysaccharide. PCR amplification showed the presence of an internal fragment of this gene in twelve strains of P. freudenreichii subsp. shermanii with a ropy phenotype in YEL+ medium. The gene sequence is highly conserved, as less than 1% of nucleotides differed among the 10 strains containing the complete gtf gene. The same primers failed to detect the gene in Propionibacterium acidipropionici strain TL 47, which is known to excrete exopolysaccharides in milk. The presence of (1-->3, 1-->2)-beta-d-glucan capsule was demonstrated for 7 out of 12 strains by agglutination with a S. pneumoniae-type 37-specific antiserum. The presence of mRNA corresponding to the gene was detected by RT-PCR in three strains at both exponential and stationary growth phases. This work represents the first identification of a polysaccharide synthase gene of P. freudenreichii, and further studies will be undertaken to elucidate the role of capsular EPS.     

Application of infrared microspectroscopy and multivariate analysis for monitoring the effect of adjunct cultures during Swiss cheese ripening

Improved cheese flavor has been attributed to the addition of adjunct cultures, which provide certain key enzymes for proteolysis and affect the dynamics of starter and nonstarter cultures. Infrared microspectroscopy provides unique fingerprint-like spectra for cheese samples and allows for rapid monitoring of cheese composition during ripening. The objective was to use infrared microspectroscopy and multivariate analysis to evaluate the effect of adjunct cultures on Swiss cheeses during ripening. Swiss cheeses, manufactured using a commercial starter culture combination and 1 of 3 adjunct Lactobacillus spp., were evaluated at d 1, 6, 30, 60, and 90 of ripening. Cheese samples (approximately 20 g) were powdered with liquid nitrogen and homogenized using water and organic solvents, and the water-soluble components were separated. A 3-μL aliquot of the extract was applied onto a reflective microscope slide, vacuum-dried, and analyzed by infrared microspectroscopy. The infrared spectra (900 to 1,800 cm⁻¹) produced specific absorption profiles that allowed for discrimination among different cheese samples. Cheeses manufactured with adjunct cultures showed more uniform and consistent spectral profiles, leading to the formation of tight clusters by pattern-recognition analysis (soft independent modeling of class analogy) as compared with cheeses with no adjuncts, which exhibited more spectral variability among replicated samples. In addition, the soft independent modeling of class analogy discriminating power indicated that cheeses were differentiated predominantly based on the band at 1,122 cm⁻¹, which was associated with S-O vibrations. The greatest changes in the chemical profile of each cheese occurred between d 6 and 30 of warm-room ripening. The band at 1,412 cm⁻¹, which was associated with acidic AA, had the greatest contribution to differentiation, indicating substantial changes in levels of proteolysis during warm-room ripening in addition to propionic acid, acetic acid, and eye formation. A high-throughput infrared microspectroscopy technique was developed that can further the understanding of biochemical changes occurring during the ripening process and provide insight into the role of adjunct nonstarter lactic acid bacteria on the complex process of flavor development in cheeses.     

Probiotic Cheddar cheese: Influence of ripening temperatures on survival of probiotic microorganisms, cheese composition and organic acid profiles

Bifidobacterium longum 1941, Bifidobacterium animalis subsp. lactis LAFTI^®B94 (B94), Lactobacillus casei 279, Lb. casei LAFTI^®L26 (L26), Lactobacillus acidophilus 4962 or Lb. acidophilus LAFTI^®L10 (L10) were used as an adjunct in the production of Cheddar cheeses which were ripened for 24 wk at 4 and 8 °C. Effects of ripening temperatures on survival of starter lactococci and probiotic microorganisms, pH and composition of cheeses and production of organic acids were examined. The counts of starter lactococci in cheeses produced with B. animalis B94, Lb. casei L26 or Lb. acidophilus 4962 ripened at 8 °C were significantly lower than those ripened at 4 °C (P < 0.05) at 24 wk. Probiotic microorganisms remained viable (>7.50 log₁₀ CFU/g) at the end of 24 wk and their viability was not affected by the ripening temperatures. There were significant effects of the type of probiotic microorganisms used, ripening time, ripening temperatures and their interactions on the concentration of lactic and acetic acids in the cheeses (P < 0.05). The acetic acid concentration in cheeses made with Bifidobacterium sp. or Lb. casei sp. was significantly higher than that of the control cheese (P < 0.05). Citric, propionic and succinic acids contents of the cheeses were not significantly affected by the type of probiotic microorganisms or ripening temperatures (P > 0.05).     

Cream cheese as a symbiotic food carrier using Bifidobacterium animalis Bb‐12 and Lactobacillus acidophilus La‐5 and inulin

The stability of cream cheeses as a symbiotic food carrier, through supplementation with different concentrations of probiotic bacteria Bifidobacterium animalis Bb‐12 and Lactobacillus acidophilus La‐5 and the prebiotic ingredient inulin was investigated. Physicochemical parameters, pH values, total solids, fat and protein levels and the viable counts of the starter lactic culture Streptococcus thermophilus and probiotic cultures, were carried out at 1, 15, 30 and 45 days of refrigerated storage (8 ± 0.5 °C). Different physicochemical characteristics were observed in all formulations. S. thermophilus showed good viability in all the trials (6.66–9.38 log cfu/g), whereas B. animalis remained above 6 log cfu/g in all the trials during the period evaluated. However, L. acidophilus showed an accentuated decline, registering values of 3.1 log cfu/g at the end of the period studied. The results suggested that cream cheese was an adequate food matrix for supplementation with probiotic bacteria, in particular B. animalis, and the prebiotic ingredient, showing potential as a symbiotic food.     

葡萄糖、乳酸钠对丙酸杆菌生长的影响

对费氏丙酸杆菌(Propionibacterium freudenreichii CS1420)发酵培养基的碳源进行优化,旨在提高菌体密度。结果表明,以乳酸钠作为单一碳源时,当乳酸钠添加量为32 g/L时,菌体生长量最大,OD600 nm为1.95;以葡萄糖作为单一碳源时,当葡萄糖添加量为8 g/L时,菌体生长量最大,OD600 nm为1.242;以乳酸钠和葡萄糖作为复合碳源时,当碳源添加量为18.8g/L乳酸钠+1.2g/L葡萄糖时,菌体生长量最大,OD600nm为2.06,这说明,复合碳源可以有效的提高菌体密度。     

Release and identification of angiotensin-converting enzyme-inhibitory peptides as influenced by ripening temperatures and probiotic adjuncts in Cheddar cheeses

The aim of the study was to examine the release of angiotensin-converting enzyme (ACE)-inhibitory peptides in Cheddar cheeses made with starter lactococci and Bifidobacterium longum 1941, B. animalis subsp. lactis LAFTI^® B94, Lactobacillus casei 279, Lb. casei LAFTI^® L26, Lb. acidophilus 4962 or Lb. acidophilusLAFTI^® L10 during ripening at 4 and 8 °C for 24 weeks. ACE-inhibitory activity of the cheeses was maximum at 24 weeks. Cheeses made with the addition of Lb. casei 279, Lb. casei LAFTI^® L26 or Lb. acidophilus LAFTI^® L10 had significantly higher (P < 0.05) ACE-inhibitory activity than those without any probiotic adjunct after 24 weeks at 4 and 8 °C. The IC₅₀ of cheeses ripened at 4 °C was not significantly different (P > 0.05) to that ripened at 8 °C. The lowest value of the IC₅₀ (0.13 mg mL⁻¹) and therefore the highest ACE-inhibitory activity corresponded to the cheese with the addition of Lb. acidophilus LAFTI^® L10. Several ACE-inhibitory peptides were identified as κ-CN (f 96–102), α_s1-CN (f 1–9), α_s1-CN (f 1–7), α_s1-CN (f 1–6), α_s1-CN (f 24–32) and β-CN (f 193–209). Most of the ACE-inhibitory peptides accumulated at the early stage of ripening, and as proteolysis proceeded, some of the peptides were hydrolyzed into smaller peptides.     

瑞士奶酪生产工艺研究

以新鲜牛乳为主要原料,根据瑞士奶酪的生产工艺,采用L9(34)正交实验的方法,研究了不同切割尺寸、发酵剂添加比例、发酵剂添加量、发酵温度和切割pH对瑞士奶酪色泽、滋气味和组织状态的影响,确定了生产瑞士奶酪的最佳工艺条件为发酵剂添加量0.02%(质量分数),凝块切割尺寸0.6cm,切割pH6.60,发酵剂(唾液链球菌嗜热亚种∶瑞士乳杆菌∶谢氏丙酸杆菌)添加比例2∶2∶1,发酵温度37℃。     

The influence of Bifidobacterium Bb-12 and lactic acid incorporation on the properties of Minas Frescal cheese

The effects of a probiotic bacterium (Bifidobacterium Bb-12) and lactic acid on the microbiological, physicochemical, rheological and microstructural proprieties of Minas Frescal cheese were evaluated after 1 day and after 28 days of storage (5 ± 1 °C). The cheese was produced with four different treatments: with no lactic acid (C1 and C2), with lactic acid (C3 and C4) and with bifidobacteria (C2 and C3). The cheese formulations containing bifidobacteria were classified as probiotic. The addition of bifidobacteria to the cheese did not influence its yield, protein or lipid levels one day after production. Moreover, after 28 days of storage, the cheese samples with no lactic acid showed lower moisture levels compared to the samples containing lactic acid (P < 0.05). The absence of lactic acid had an influence on the rheological and microstructural behavior, making them more elastic, firm and compact, however all the cheese samples evaluated showed a higher tendency to viscosity than to elasticity. The Minas Frescal cheese with Bifidobacterium Bb-12 and lactic acid showed great potential as a functional food, with possible industrial and commercial application.