The ecology of non-starter lactic acid bacteria (NSLAB) and their use as adjuncts in New Zealand Cheddar

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Abstract

This paper reviews the recent New Zealand studies on non-starter lactic acid bacteria (NSLAB) in Cheddar cheese. In New Zealand, cheese adventitious NSLAB were usually strains of homofermentative lactobacilli and occasionally pediococci and heterofermentative lactobacilli. In Cheddar cheese, manufactured in six factories at different times and ripened over 24 months, over 140 NSLAB strains were identified using pulse field gel electrophoresis profiles and fermentation patterns on 22 carbohydrates. The majority of NSLAB isolates identified belong to two species: Lactobacillus paracasei subsp. paracasei and Lactobacillus rhamnosus. The composition of the NSLAB strains in a cheese was variable and dependent on the manufacturing factory, the date of manufacture and the age of the cheese. In most cheeses, no single NSLAB strain predominated during ripening. Up to six strains formed the majority (about 90%) of the NSLAB population, the remainder was represented by up to 15 other strains. In only about 10% of the cheeses investigated was one strain predominant throughout the 24 month ripening period. The acid and salt tolerance, effect of temperature, ability to produce biogenic amines and the lipolytic and proteolytic activities of the NSLAB strains were studied to screen strains for use as adjuncts. Sixty suitable strains were selected for further screening in a cheese model system. The NSLAB were incubated at 30°C for 10 days in the model system and the flavour and biochemical changes were assessed. Twenty-three strains were eliminated as potential adjuncts because they produced defective flavours and a further 13 strains were eliminated on the basis of undesirable biochemical properties (e.g. excess fermentation of glutamate, lactate racemisation). The 24 remaining NSLAB strains that had acceptable microbiological biochemical and flavour properties were used as adjuncts in Cheddar trials. Seven of the 24 adjunct combinations studied (made up from 2–4 strains of NSLAB) provided consistent flavour benefits. Analysis of the cheeses during ripening in these trials provided significant understanding of the ripening mechanisms of the NSLAB adjuncts.

Introduction

The non-starter lactic acid bacteria (NSLAB) present in ripened Cheddar cheese originate either from the factory environment or the milk as adventitious contaminants (Naylor & Sharpe, 1958; Law, Castañon, & Sharpe, 1976; Peterson & Marshall, 1990; Martley & Crow, 1993). The NSLAB can be isolated from cheese and enumerated using Selective Rogosa Agar (Rogosa, Mitchell, & Wiseman, 1951; Franklin & Sharpe, 1963). The types of NSLAB identified include homofermentative and heterofermentative mesophilic lactobacilli and pediococci.

Cheddar cheese flavour can develop in the absence of NSLAB as illustrated with the use of aseptic cheese vats and specific starter strains, but these cheeses are considered to lack the full mature flavour (Perry & McGillivray, 1964; Reiter et al., 1967). The specific ripening mechanisms of NSLAB that contribute positively to Cheddar flavour have not been determined (Martley & Crow, 1993) due to the complex balance of components that constitutes Cheddar flavour and the problem of differentiating the ripening contributions of NSLAB from those of other components (e.g. starters, rennet, milk enzymes and chemical reactions). Some Cheddar defects, however, are attributable to specific metabolic activities of NSLAB. For example, the formation of white spots of calcium lactate crystals in ripened Cheddar is due to the racemisation of l(+)-lactate to an isomer mixture of lactate by certain NSLAB strains (Thomas & Crow, 1983). Similarly, slits in Cheddar have been attributed to heterofermentative lactobacilli (Laleye, Simard, Lee, Holley, & Giroux, 1987).

The apparently random nature of the flavour contribution by NSLAB to Cheddar ripening was exemplified by an early report by Sherwood (1939) who identified four groups of Lb. plantarum in New Zealand cheese: a group that provided beneficial qualities to Cheddar cheese, a group that appeared to have no effect and two groups that produced serious defects.

The deliberate addition of NSLAB to cheesemilk as adjuncts for Cheddar flavour improvement has had varying success (Peterson & Marshall, 1990). An early study by Lane and Hammer (1935) found that different strains of Lb. casei generally improved flavour over the control cheese, though there were differences in the flavour profile between strains. More recently, different groups have further investigated the use of a variety of species and strains of NSLAB as adjuncts (Puchades, Lemieux, & Simard, 1989; Broome, Krause, & Hickey, 1990; Lee, Laleye, Simard, Munsch, & Holley, 1990; McSweeney et al., 1994; Lane & Fox, 1996). In many studies both flavour quality and intensity was improved with the use of NSLAB adjuncts. Two strains of Lb. paracasei that have probiotic potential can also be effectively delivered in Cheddar via adjuncts (Gardiner, Ross, Collins, Fitzgerald, & Stanton, 1998).

Successful NSLAB adjuncts require two important features. The first is that the strains must provide a balance of beneficial ripening reactions in cheese. As noted previously, this balance is poorly understood and at best adjuncts are selected on a few positive features—but more typically adjuncts are selected on the basis of the absence of specific defects. The second consideration is that adjunct strains need to be competitive against the adventitious NSLAB in the Cheddar cheese and remain the dominant NSLAB during ripening to effect their flavour benefits. In many cases, this feature is obtained by selecting NSLAB isolates from good quality Cheddar.

This means that the species and strains best suited to be used as NSLAB adjuncts could vary between countries and even between factories within a country. This choice would reflect the different adventitious NSLAB dominant in both the manufacturing and ripening environments for Cheddar. Differences in the Cheddar ripening environment such as pH, salt-in-moisture and the ripening temperature profile will be important in determining which of the strains initially present at low densities in the young curd will become the dominant strains.

The most common NSLAB species found in Irish Cheddar cheese were Lb. casei, Lb. plantarum and Lb. curvatus (Jordan & Cogan, 1993). In New Zealand, the dominant species in Cheddar cheese manufactured in a large number of factories in the 1930s was Lb. plantarum (Sherwood, 1939). However, in the Cheddar cheese from New Zealand in the 1990s the major two species of NSLAB were Lb. paracasei and Lb. rhamnosus (for data refer to results given later in this paper). The reasons why the dominant NSLAB species in New Zealand Cheddar were different in the 1930s and in the 1990s are not known. The manufacturing practices employed today in the large cheese plant, particularly the higher hygiene standard, are probably important factors.

In this paper, the recent work on NSLAB in New Zealand Cheddar is reviewed, illustrated by some as yet unpublished data. The ecology of NSLAB, the reasons for using NSLAB adjuncts, the selection procedures for test adjuncts and the results of using NSLAB adjuncts in Cheddar are discussed.

Section snippets

Ecology of NSLAB in New Zealand Cheddar

Cheddar cheeses from three New Zealand cheese factories manufactured each month over the 1993/1994 and 1994/1995 seasons were ripened at 13°C. The cheeses were analysed over 24 months for the densities and composition of NSLAB as well as for biochemical and sensory changes. The composition of the cheeses was in the range typical of New Zealand Cheddars (pH 5.1–5.3; salt in moisture 4.9–5.3%). NSLAB were isolated on Rogosa agar (pH adjusted to 5.35±0.05) incubated anaerobically for 5 days at

The benefits of NSLAB adjuncts to New Zealand Cheddar manufacturers

Many of the variables in the cheese making processes in modern New Zealand factories are under good control. The milk is standardised and pasteurised, defined starter cultures are used and the rennet has defined activity. There is good hygiene. Cooking temperature, run and salt pH, times in the vat and on the belt, curd particle size and salt addition rate, are all under tight control. The result is Cheddar cheese of defined composition. Such cheeses could be expected to develop a consistent

Selection of NSLAB strains for adjunct cheese trials

Over 140 strains of NSLAB were identified in the New Zealand Cheddars studied. It was not practicable to use all these strains in cheese trials, particularly if combinations of strains were required. Single strains that form the predominant NSLAB throughout ripening of good quality Cheddar were assumed to be good candidates as adjuncts. This occurred in only about 10% of the cheeses monitored, but provided two candidate strains of Lb. rhamnosus and six of Lb. paracasei. The majority of the 140

Use of NSLAB adjuncts in Cheddar trials

Using the 24 selected NSLAB strains, 20 adjunct combinations (made up from 2 to 4 strains at equal densities) were tested in Cheddar cheese trials under typical New Zealand conditions. The adjunct combinations were added to give 300–1000 cfu/mL of vat milk.

During ripening, one adjunct combination did not show any improvement compared with a typical control cheese, 12 adjunct combinations showed some flavour improvements and seven adjunct combinations showed significant flavour improvements.

The

Summary

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    NSLAB adjuncts are required for improved flavour control in aged cheeses such as mature Cheddar as uncontrolled adventitious strains of NSLAB can cause defects.

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    In New Zealand Cheddar the majority of adventitious NSLAB strains belong to two species, Lb. paracasei and Lb. rhamnosus. The composition of adventitious NSLAB in cheeses varies between factories and between days of manufacture. The composition in a cheese also changes during ripening.

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    Microbiological and biochemical features supported by

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